Pathogenic escherichia coli associated protein

ABSTRACT

The present invention provides a polypeptide, called EspA, which is secreted by pathogenic  E. coli,  such as the enteropathogenic (EPEC) and enterohemorrhagic (EHEC) E. coli. The invention also provides isolated nucleic acid sequences encoding EspA polypeptide, EspA peptides, a recombinant method for producing recombinant EspA, antibodies which bind to EspA, and a kit for the detection of EspA-producing  E. coli .

CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims priority from U.S. ProvisionalApplication No. 60/015,999, filed Apr. 23, 1996.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with support from Public Health Serviceaward AI32074 from the National Institutes of Health. The United Statesgovernment may have certain rights in this invention.

FIELD OF THE INVENTION

[0003] The present invention relates generally to the virulence ofpathogenic organisms and more specifically to virulence factorsassociated with enteropathogenic bacteria.

BACKGROUND OF THE INVENTION

[0004] Antibiotics have been used for years to successfully treatdiverse bacterial infections. However, bacterial resistance toantibiotics has been an increasing problem over the past few years. Manypathogens are now resistant to several antibiotics, and in some cases,the diseases they cause are no longer treatable with conventionalantibiotics. Despite the past successes of antibiotics, there have beenfew, if any, new classes of antibiotics developed in the past twodecades. New variations on existing drugs have been introduced, butresistance to these compounds usually arises within a short period oftime.

[0005] Many studies have shown that if a mutation is made in a gene thatencodes a virulence factor, the organism containing that gene is nolonger pathogenic. Additionally, if a host is vaccinated against avirulence factor, disease can often be blocked. However, it has not beenshown that specific inhibition of a virulence factor can attenuatedisease.

[0006] The mechanisms of action for toxins, adherence, invasion,intracellular parasitism, have been studied. However, each virulencefactor uses a different mechanism, which has made the development of abroad spectrum inhibitor impossible. One conserved factor that could beconsidered a target for a therapeutic is a two-component regulatorysystem. However, this system is not specific for virulence factors, andis used in several bacterial housekeeping systems. Additionally, thesystems have been identified in eukaryotic systems, which would increasethe risk of host toxicity if an inhibitor was utilized. To develop anideal anti-infective agent, the bacterial virulence mechanism that theantibiotic affects should be universal for many pathogens, specific forvirulence mechanisms, and not be present in host cells. One such systemthat has recently been identified is the bacterial type III secretionsystem.

[0007] Gram-negative bacteria utilize specialized machinery to exportmolecules across their two membranes and the piroplasm, a processcritical for moving virulence factors to the bacterial surface wherethey can interact with host components. Gram-negative secretion has beendivided into four major pathways. First, the Type I secretion is used bya small family of toxins, with E. coli hemolysin being the prototype.Second, the type II secretion system is the major export pathway used bymost Gram-negative bacteria to export many molecules, including somevirulence factors; it shares homology to mammalian drug resistancemechanisms. Third, the type IV secretion system is encoded within thesecreted product, which cleaves itself as part of the secretionmechanism; the prototype of this system is the Neisseria IgA protease.Fourth, the most recently discovered secretion pathway, is the type IIIpathway.

[0008] Type III secretion systems were originally described as asecretion system for Yersinia secreted virulence proteins, YOPs, whichare critical for Yersinia virulence. A homologous secretion system wasthen identified in several plant pathogens, including Pseudomonassyringae, P. solanacearurn, and Xantharnonas carnpestris. These plantpathogens use this secretion pathway to secrete virulence factors(harpins and others) that are required for causing disease in plants.Although the secretion system is similar, harpins and YOPs (i.e. thesecreted virulence factors) are not homologous polypeptides. Severalother type III secretion systems necessary for virulence have morerecently been identified in other pathogens. These systems include theinvasion systems Salmonella and Shigella use to enter cells and causedisease. Another type III secretion system has been identified inSalmonella which is critical for disease, although the secreted productsof this pathway and the virulence mechanisms have not been establishedyet. Pseudomonas aeruginosa has a type III secretion system necessaryfor secretion of Exoenzyme S, a potent virulence factor.

[0009] Enteropathogenic Escherichia coli (EPEC) is a leading cause ofinfant diarrhea and was the first E. coli shown to causegastroenteritis. Enteropathogenic E. coli activates the host epithelialcells' signal transduction pathways and causes is cytoskeletalrearrangement, along with pedestal and attaching/effacing lesionformation.

[0010] A three-stage model has describes enteropathogenic E. colipathogenesis. An initial localized adherence to epithelial cells,mediated by a type IV fimbria, is followed by the activation of hostepithelial cell signal transduction pathways and intimate attachment tohost epithelial cells. These final two steps are collectively known asattaching and effacing. The signal transduction in the host epithelialcells involves activation of host cell tyrosine kinase activity leadingto tyrosine phosphorylation of a 90 kilodalton host membrane protein,Hp9.0, and fluxes of intracellular inositol phosphate (IP₃) and calcium.Following this signal transduction, the bacteria adheres intimately tothe surface of the epithelial cell, accompanied by damage to hostepithelial cell microvilli and accumulation of cytoskeletal proteinsbeneath the bacteria.

SUMMARY OF THE INVENTION

[0011] The present invention is based on the discovery a proteinassociated with virulence in pathogenic bacteria, for exampleenteropathogenic E. coli.

[0012] DNA sequence analysis of the Locus of Enterocyte Effacementbetween eaeA and espB identified a gene (espA) that matched the aminoterminal sequence of the 25 kilodalton enteropathogenic E. coli secretedprotein. A mutant with an insertion in espA does not secrete thisprotein, activate epithelial cell signal transduction or causecytoskeletal rearrangement. However, these functions could becomplemented by a cloned wild-type espA gene.

[0013] Two enteropathogenic E. coli genes, espA and espB, that encodesecreted virulence factors, EspA and EspB respectively, were cloned andsequenced. These proteins were shown to be involved in the triggering ofhost epithelial signal transduction pathways and invasion. Since EspA isa secreted protein, it is ideally suited for use in a fusion proteinlinked to a polypeptide of interest.

[0014] The type III secretion pathway is an ideal target for potentialinhibitors, because it is a virulence factor-specific conserved pathwayidentified in bacteria. The present invention provides a method foridentifying inhibitors of type III secretion systems. The relevance ofthis invention is directed toward the development of new antibacterialtherapeutics. In contrast to other antibiotics, the compounds identifiedby the method of this invention will not kill or inhibit growth ofpathogens; instead, the compounds will block the secretion of virulencefactors that are critical to causing disease. Because the type IIIsecretion system is the first virulence mechanism that shows a largedegree of conservation between diverse pathogens, some of the compoundsidentified by the method of this invention will be broad spectrumtherapeutics. The benefit would be the identification of newtherapeutics that could be used in the treatment of several human,animal, and plant diseases.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015]FIG. 1 shows the nucleotide and deduced amino acid sequence ofespA (herein referred to as SEQ ID NO: 1 and SEQ ID NO:2, respectively).Potential ribosome binding sites are underlined. Nucleotides included inprimers Donne-99 and Donne-100, flanking the deletion engineered inpLCL121, are shaded.

[0016]FIG. 2 shows the construction of a non-polar mutation in espA.Primers Donne-90 and the reverse primer were used to amplify a fragmentcontaining a 5′ portion of the gene, which was cloned into pCRscript tocreate pLCL119. Primers Donne-100 and the universal primer were used toamplify a fragment containing a 3′ portion of the gene, which was clonedinto pCRscript to create pLCL120. New NruI sites were incorporated intoboth Donne-99 and Donne-100 so that the NruI-SalI fragment of pLCL120could be cloned into pLCL119 to create pLCL121, which has a 150 bpdeletion within the espA gene. A SmaI fragment from pUC18K containingthe aphA-3 kanamycin resistance gene was cloned into the NruI site ofpLCL121 to create pLCL122. This insertion results in a transcriptionalfusion of the aphA-3 gene and a translational fusion of the 3′ end ofthe espA gene, with preservation of the espA reading frame. The ribosomebinding sites are underlined.

[0017]FIG. 3 shows a genetic map of the plasmids containing RDEC-1 (A)and enteropathogenic Escherichia coli (EPEC) (B) espA, espD, and espBgenes. Arrows indicate positions that stop codon insertions were made inespA and espB (A), and the frame shift mutation engineered into theBglII site in espD (B). The 250 base pair deletion in espB is marked by//. Solid and clear boxes represent open reading frames and predictedopen reading frames. Restriction enzymes are indicated as follows: Bam,BamHI; Ec, EcoRI; Bg, BglII; Xb, XbaI; Sa, SalI.

[0018]FIG. 4 shows the nucleotide sequence of RDEC-1 espA (A) (hereinreferred to as SEQ ID NO: 3 and SEQ ID NO:4, respectively) and espB (B)(herein referred to as SEQ ID NO: 5 and SEQ ID NO:6, respectively).Asterisks indicate stop codons. Potential ribosome binding sites areunderlined. Predicted amino acid sequences of EspA and EspB are alignedin C) (SEQ ID NO:7-14). Shaded areas represent identity. Nucleotide andamino acid sequences have been deposited into the EMBL GenBank and theiraccession numbers are as follows: RDEC-1 espA (U80908), RDEC-1 espB(U80796), enteropathogenic Escherichia coli strain E2348/69 espA(Z54352), enteropathogenic Escherichia coli strain E2348/69 espB(Z21555), enteropathogenic Escherichia coli strain E2348/69 espD(Y09228), enterohemorrhagic Escherichia coli strain EDL933 serotype 0157espB (X96953), enterohemorrhagic Escherichia coli strain 413/89-1serotype 026 espb (X99670).

DETAILED DESCRIPTION

[0019] The present invention provides a polypeptide, called EspA, whichis secreted by pathogenic E. coli, such as the enteropathogenic (EPEC)and enterohemorrhagic (EHEC) E. coli. Diagnosis of disease caused bysuch pathogenic E. coli can be performed by standard techniques, such asthose based upon the use of antibodies which bind to EspA to detect theprotein, as well as those based on the use of nucleic acid probes fordetection of nucleic acids encoding EspA polypeptide. The invention alsoprovides isolated nucleic acid sequences encoding EspA polypeptide, EspApeptides, a recombinant method for producing recombinant EspA,antibodies which bind to EspA, and a kit for the detection ofEspA-producing E. coli. The invention also provides a method ofimmunizing a host with EspA to induce a protective immune response toEspA.

[0020] The details of the preferred embodiments of the present inventionare set forth in the accompanying drawings and the description below.Once the details of the

[0021] As used herein, the term “EspA” (for EPEC secreted [or signaling]protein A) refers to a polypeptide which is a secreted protein fromenteropathogenic or enterohemorrhagic E. coli and has a molecular weightof about 25 kilodaltons as determined by SDS-PAGE. EspA is anenteropathogenic E. coli-secreted protein necessary for activatingepithelial cell signal transduction, intimate contact, and formation ofattaching and effacing lesions, processes correlated with disease. Anexample of epithelial cells are cells.

[0022] As used herein, the term “polypeptide” encompasses any naturallyoccurring allelic variant thereof as well as manufactured recombinantforms. As used herein, EspA polypeptides encompass both naturallyoccurring and recombinant forms, i.e., non-naturally occurring forms ofthe protein and the peptide that are sufficiently identical to naturallyoccurring EspA peptide to have a similar function of causingpathogenicity. Examples of such polypeptides include the EspApolypeptides from enteropathogenic and enterohemorrhagic E. coli, butare not limited to them. Protein and polypeptides include derivatives,analogs and peptidomimetics. Alternatively, EspA peptides can bechemically synthesized using synthesis procedures known to one skilledin the art. Preferably, an automated peptide synthesizer is used withN^(α)Fmoc amino acids on a polyethylene glycol-polystyrene (PEGPS) graftresin. Suitable linkers such as a peptide amide linker (PAL) can beused, for example, to create carboxamide end groups.

[0023] The term “substantially pure” is used herein to describe amolecule, such as a polypeptide (e.g., an EspA polypeptide, or afragment thereof) that is substantially free of other proteins, lipids,carbohydrates, nucleic acids, and other biological materials with whichit is naturally associated. For example, a substantially pure molecule,such as a polypeptide, can be at least 60%, by dry weight, the moleculeof interest. One skilled in the art can purify EspA polypeptides usingstandard protein purification methods and the purity of the polypeptidescan be determined using standard methods including, e.g., polyacrylamidegel electrophoresis (e.g., SDS-PAGE), column chromatography (e.g., highperformance liquid chromatography (HPLC)), and amino-terminal amino acidsequence analysis.

[0024] EspA polypeptides included in the invention can have one of theamino acid sequences of EspAs from human or rabbit enteropathogenic E.coli, for example, the amino acid sequence of FIG. 1 or FIG. 4. EspApolypeptides, such as those shown in FIGS. 1 and 4, can be characterizedby being approximately 25 kD as determined by SDS-PAGE.

[0025] Also included in the invention are polypeptides having sequencesthat are “substantially identical” to the sequence of an EspApolypeptide, such as one of EspAs in FIGS. 1 and 4. A “substantiallyidentical” amino acid sequence is a sequence that differs from areference sequence only by conservative amino acid substitutions, forexample, substitutions of one amino acid for another of the same class(e.g., substitution of one hydrophobic amino acid, such as isoleucine,valine, leucine, or methionine, for another, or substitution of onepolar amino acid for another, such as substitution of arginine forlysine, glutamic acid for aspartic acid, or glutamine for asparagine),or by one or more non-conservative substitutions, deletions, orinsertions, provided that the polypeptide retains at least oneEspA-specific activity or an EspA-specific epitope. For example, one ormore amino acids can be deleted from an EspA polypeptide, resulting inmodification of the structure of the polypeptide, without significantlyaltering its biological activity. For example, amino- orcarboxyl-terminal amino acids that are not required for EspA biologicalactivity, can be removed. Such modifications can result in thedevelopment of smaller active EspA polypeptides.

[0026] Other EspA polypeptides included in the invention arepolypeptides having amino acid sequences that are at least 50% identicalto the amino acid sequence of an EspA polypeptide, such as any of EspAsin FIGS. 1 and 4. The length of comparison in determining amino acidsequence homology can be, for example, at least 15 amino acids, forexample, at least 20, 25, or 35 amino acids. Homology can be measuredusing standard sequence analysis software (e.g., Sequence AnalysisSoftware Package of the Genetics Computer Group, University of WisconsinBiotechnology Center, 1710 University Avenue, Madison, Wis. 53705; alsosee Ausubel, et al., supra).

[0027] The invention also includes fragments of EspA polypeptides thatretain at least one EspA-specific activity or epitope. For example, anEspA polypeptide fragment containing, e.g., at least 8-10 amino acidscan be used as an immunogen in the production of EspA-specificantibodies. The fragment can contain, for example, an amino acidsequence that is conserved in EspAs. In addition to their use as peptideimmunogens, the above-described EspA fragments can be used inimmunoassays, such as ELISAs, to detect the presence of EspA-specificantibodies in samples.

[0028] The EspA polypeptides of the invention can be obtained using anyof several standard methods. For example, EspA polypeptides can beproduced in a standard recombinant expression systems (see below),chemically synthesized (this approach may be limited to small EspApeptide fragments), or purified from tissues in which they are naturallyexpressed (see, e.g., Ausubel, et al., supra).

[0029] The invention also provides isolated nucleic acid molecules thatencode the EspA polypeptides described above, as well as fragmentsthereof. For example, nucleic acids that encode EspAs as in FIGS. 1 and4 are included in the invention. These nucleic acids can containnaturally occurring nucleotide sequences (see FIGS. 1 and 4), orsequences that differ from those of the naturally occurring nucleicacids that encode EspAs, but encode the same amino acids, due to thedegeneracy of the genetic code. The nucleic acids of the invention cancontain DNA or RNA nucleotides, or combinations or modificationsthereof.

[0030] By “isolated nucleic acid” is meant a nucleic acid, e.g., a DNAor RNA molecule, that is not immediately contiguous with the 5′ and 3′flanking sequences with which it normally is immediately contiguous whenpresent in the naturally occurring genome of the organism from which itis derived. The term thus describes, for example, a nucleic acid that isincorporated into a vector, such as a plasmid or viral vector; a nucleicacid that is incorporated into the genome of a heterologous cell (or thegenome of a homologous cell, but at a site different from that at whichit naturally occurs); and a nucleic acid that exists as a separatemolecule, e.g., a DNA fragment produced by PCR amplification orrestriction enzyme digestion, or an RNA molecule produced by in vitrotranscription. The term also describes a recombinant nucleic acid thatforms part of a hybrid gene encoding additional polypeptide sequencesthat can be used, for example, in the production of a fusion protein.

[0031] The nucleic acid molecules of the invention can be used astemplates in standard methods for production of EspA gene products(e.g., EspA RNAs and EspA polypeptides; see below). In addition, thenucleic acid molecules that encode EspA polypeptides (and fragmentsthereof) and related nucleic acids, such as (1) nucleic acids containingsequences that are complementary to, or that hybridize to, nucleic acidsencoding EspA polypeptides, or fragments thereof (e.g., fragmentscontaining at least 12, 15, 20, or 25 nucleotides); and (2) nucleicacids containing sequences that hybridize to sequences that arecomplementary to nucleic acids encoding EspA polypeptides, or fragmentsthereof (e.g., fragments containing at least 12, 15, 20, or 25nucleotides); can be used in methods focused on their hybridizationproperties. For example, as is described in further detail below, suchnucleic acid molecules can be used in the following methods: PCR methodsfor synthesizing EspA nucleic acids, methods for detecting the presenceof an EspA nucleic acid in a sample, screening methods for identifyingnucleic acids encoding new EspA family members, and therapeutic methods.

[0032] The invention also includes methods for identifying nucleic acidmolecules that encode members of the EspA polypeptide family in additionto EspAs shown in FIGS. 1 and 4. In these methods, a sample, e.g., anucleic acid library, such as a cDNA library, that contains a nucleicacid encoding an EspA polypeptide is screened with an EspA-specificprobe, e.g., an EspA-specific nucleic acid probe. EspA-specific nucleicacid probes are nucleic acid molecules (e.g., molecules containing DNAor RNA nucleotides, or combinations or modifications thereof) thatspecifically hybridize to nucleic acids encoding EspA polypeptides, orto complementary sequences thereof. The term “EspA-specific probe,” inthe context of this method of invention, refers to probes that bind tonucleic acids encoding EspA polypeptides, or to complementary sequencesthereof, to a detectably greater extent than to nucleic acids encodingother polypeptides, or to complementary sequences thereof. The term“EspA-specific probe” thus includes probes that can bind to nucleicacids encoding EspA polypeptides (or to complementary sequencesthereof).

[0033] The invention facilitates production of EspA-specific nucleicacid probes. Methods for obtaining such probes can be designed based onthe amino acid sequence alignments shown in FIGS. 1-3. The probes, whichcan contain at least 12, e.g., at least 15, 25, 35, 50, 100, or 150nucleotides, can be produced using any of several standard methods (see,e.g. Ausubel, et al., supra). For example, preferably, the probes aregenerated using PCR amplification methods. In these methods, primers aredesigned that correspond to EspA-conserved sequences, which can includeEspA-specific amino acids, and the resulting PCR product is used as aprobe to screen a nucleic acid library, such as a cDNA library. Anucleotide sequence encoding EspA was identified generally followingthis process based upon the analysis of the sequences of EspA in FIGS. 1and 4.

[0034] As is known in the art, PCR primers are typically designed tocontain at least 15 nucleotides, for example 15-30 nucleotides. Thedesign of EspA-specific primers containing 21 nucleotides, which encodeEspA peptides containing 7 amino acids, are described as follows.Preferably, most or all of the nucleotides in such a probe encodeEspA-conserved amino acids, including EspA-specific amino acids. Forexample, primers containing sequences encoding peptides containing atleast 40% EspA-conserved amino acids can be used. Such a primer,containing 21 nucleotides, can include sequences encoding at least 3EspA-conserved amino acids. Thus, the primer can contain sequencesencoding at least one EspA-specific amino acid, for example, up to 7EspA-specific amino acids. Once EspA-specific amino acid sequences areselected as templates against which primer sequences are to be designed,the primers can be synthesized using, e.g., standard chemical methods.As is described above, due to the degeneracy of the genetic code, suchprimers should be designed to include appropriate degenerate sequences,as can readily be determined by one skilled in the art.

[0035] As used herein, the term “espA” refers to polynucleotide encodingthe EspA polypeptide. These polynucleotides include DNA, cDNA and RNAsequences which encode EspA. All polynucleotides encoding all or aportion of EspA are also included herein. Such polynucleotides includenaturally occurring, synthetic, and intentionally manipulatedpolynucleotides. For example, a espA polynucleotide can be subjected tosite-directed mutagenesis. The espA polynucleotide sequence alsoincludes antisense sequences. All degenerate nucleotide sequences areincluded in the invention as long as the amino acid sequence of EspApeptide encoded by the nucleotide sequence is functionally unchanged.

[0036] This invention encompasses nucleic acid molecules that hybridizeto the polynucleotide of the invention. As used herein, the term“nucleic acid” encompasses RNA as well as single and double-stranded DNAand cDNA. The polynucleotide encoding EspA includes the nucleotidesequence in FIGS. 1 and 4, as well as nucleic acid sequencescomplementary to that sequence. A complementary sequence may include anantisense nucleotide. When the sequence is RNA, the deoxynucleotides A,G, C, and T of FIGS. 1 and 4 are replaced by ribonucleotides A, G, C,and U, respectively. Also included in the invention are fragments of theabove-described nucleic acid sequences that are at least 15 bases inlength, which is sufficient to permit the fragment to selectivelyhybridize to DNA that encodes the protein of FIG. 1 or 4 underphysiological conditions.

[0037] In nucleic acid hybridization reactions, the conditions used toachieve a particular level of stringency will vary, depending on thenature of the nucleic acids being hybridized. For example, the length,degree of complementarity, nucleotide sequence composition (e.g., GC v.AT content), and nucleic acid type (e.g., RNA v. DNA) of the hybridizingregions of the nucleic acids can be considered in selectinghybridization conditions. An additional consideration is whether one ofthe nucleic acids is immobilized, for example, on a filter.

[0038] An example of progressively higher stringency conditions is asfollows: 2× SSC/0.1% SDS at about room temperature hybridizationconditions); 0.2× SSC/0.1% SDS at about room temperature (low stringencyconditions); 0.2× SSC/0.1% SDS at about 42° C. (moderate stringencyconditions); and 0.1× SSC at about 68° C. high stringency conditions).Washing can be carried out using only one of these conditions, e.g.,high stringency conditions, or each of the conditions can be used, e.g.,for 10-15 minutes each, in the order listed above, repeating any or allof the steps listed. However, as mentioned above, optimal conditionswill vary, depending on the particular hybridization reaction involved,and can be determined empirically.

[0039] DNA sequences of the invention can be obtained by severalmethods. For example, the DNA can be isolated using hybridizationtechniques which are well known in the art. These include, but are notlimited to (1) hybridization of libraries with probes to detecthomologous nucleotide sequences, (2) polymerase chain reaction (PCR) onDNA using primers capable of annealing to the DNA sequence of interest,and (3) antibody screening of expression libraries to detect cloned DNAfragments with shared structural features.

[0040] Screening procedures which rely on nucleic acid hybridizationmake it possible to isolate any gene sequence from any organism,provided the appropriate probe is available. Oligonucleotide probes,which correspond to a part of the sequence encoding the protein inquestion, can be synthesized chemically or produced by fragmentation ofthe native sequence. Chemical synthesis requires that short,oligopeptide stretches of amino acid sequence be known. The DNA sequenceencoding the protein can be deduced from the genetic code, however, thedegeneracy of the code must be taken into account. It is possible toperform a mixed addition reaction when the sequence is degenerate. Thisincludes a heterogeneous mixture of denatured double-stranded DNA. Forsuch screening, hybridization is preferably performed on eithersingle-stranded DNA or denatured double-stranded DNA. When used incombination with polymerase chain reaction technology, even rareexpression products can be cloned.

[0041] The invention provides nucleic acid sequences encoding the EspApolypeptides, vectors and host cells containing them and methods ofexpression. After a peptide of EspA is isolated, nucleic acids encodingthe peptide can be isolated by methods well known in the art. Theseisolated nucleic acids can be ligated into vectors and introduced intosuitable host cells for expression. Methods of ligation and expressionof nucleic acids within cells are well known in the art (see, Sambrooket al., Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y., 1989, incorporated herein byreference).

[0042] As used herein, the terms “espB” and “eaeA” refer to genes otherthan espA that encode enteropathogenic E. coli-secreted proteins. Asused herein, the term “EspB” and “EaeA” refer to the proteins encoded bythe espB and the eaeA genes, respectively.

[0043] The invention provides vectors containing polynucleotidesencoding the EspA polypeptide. For example, the plasmid (pMSD2) with anintact espA can restore secretion of the EspA protein in an espAdeficient strain. As used herein, “vectors” includes plasmids, DNA andRNA viral vectors, baculoviral vectors, vectors for use in yeast, andother vectors well known to those of skill in the art. Several types ofvectors are commercially available and can be used to practice thisinvention. Examples of vectors useful in the practice of this inventioninclude those as widely varied as the low-copy vector pMW118, thepositive-selection suicide vector pCVD442, and the commerciallyavailable pBluescript II SK(+) (Stragene, La Jolla, Calif.).

[0044] When the vector is a plasmid, it generally contains a variety ofcomponents including promoters, signal sequences, phenotypic selectiongenes, origins of replication sites, and other necessary components asare known to those of skill in the art. Promoters most commonly used inprokaryotic vectors include the lacZ promoter system, the alkalinephosphatase pho A promoter, the bacteriophage λPL promoter (atemperature sensitive promotor), the tac promoter (a hybrid trp-lacpromoter regulated by the lac repressor), the tryptophan promoter, andthe bacteriophage T7 promoter. For example, the low-copy vector pMW118under control of the lacZ promoter.

[0045] A signal sequence is typically found immediately 5′ to thenucleic acid encoding the peptide, and will thus be transcribed at theamino terminus of the fusion protein.

[0046] Typical phenotypic selection genes are those encoding proteinsthat confer antibiotic resistance upon the host cell. For example,ampicillin resistance gene (amp) and the tetracycline resistance gene(tet) are readily employed for this purpose. For a different example,the aphA-3 cassette, encoding a gene for resistance to kanamycin (kan),may be cloned into the region of vector containing polynucleotidesencoding the EspA polypeptide for selection of the vector on kanamycinplates.

[0047] Construction of suitable vectors containing polynucleotidesencoding EspA polypeptide are prepared using standard recombinant DNAprocedures well known to those of skill in the art. Isolatedpolynucleotides encoding the EspA polypeptide to be combined to form thevector are cleaved and ligated together in a specific order andorientation to generate the desired vector.

[0048] The invention provides a host cell containing a vector having apolynucleotide encoding the EspA polypeptide. The polynucleotides of thepresent invention can be used to produce transformed or transfectedcells for enhanced production of the expressed EspA. EspA can beisolated from transformed cells by standard methods well known to thoseof skill in the art. The protein could be isolated, for example, usingimmunoaffinity purification.

[0049] DNA sequences encoding EspA can be expressed in vitro by DNAtransfer into a suitable host cell. “Host cells” are cells in which avector can be propagated and its DNA expressed. The term also includesany progeny of the subject host cell. It is understood that all progenymay not be identical to the parental cell since there may be mutationsthat occur during replication. However, such progeny are included whenthe term “host cell” is used. Methods of stable transfer, meaning thatthe foreign DNA is continuously maintained in the host, are known in theart.

[0050] In the present invention, the EspA polynucleotide sequences maybe inserted into a recombinant expression vector. The term “recombinantexpression vector” refers to a plasmid, virus or other vehicle known inthe art that has been manipulated by insertion or incorporation of theEspA genetic sequences. Such expression vectors contain a promotersequence which facilitates the efficient transcription of the insertedgenetic sequence of the host. The expression vector typically containsan origin of replication, a promoter, as well as specific genes whichallow phenotypic selection of the transformed cells.

[0051] Polynucleotide sequences encoding EspA can be expressed in eitherprokaryotes or eukaryotes. Hosts can include microbial, yeast, insectand mammalian organisms. Methods of expressing DNA sequences havingeukaryotic or viral sequences in prokaryotes are well known in the art.Biologically functional viral and plasmid DNA vectors capable ofexpression and replication in a host are known in the art. Such vectorsare used to incorporate DNA sequences of the invention.

[0052] Transformation of a host cell with recombinant DNA may be carriedout by conventional techniques as are well known to those skilled in theart. Where the host is prokaryotic, such as E. coli, competent cellswhich are capable of DNA uptake can be prepared from cells harvestedafter exponential growth phase and subsequently treated by the CaCl₂method using procedures well known in the art. Alternatively, MgCl₂ orRbCl can be used. Transformation can also be performed after forming aprotoplast of the host cell if desired. For another example, triparentalconjugation may be used to genetically introduce vector into E. coli,especially enteropathogenic E. coli or rabbit enteropathogenic E. coli.The transformed cells are selected by growth on an antibiotic, commonlytetracycline (tet) or ampicillin (amp), to which they are renderedresistant due to the presence of tet or amp resistance genes on thevector. In a specific embodiment, cells are selected on the basis ofresistance to kanamycin and sucrose.

[0053] When the host is a eukaryote, such methods of transfection of DNAas calcium phosphate co-precipitates, conventional mechanical proceduressuch as micro-injection, electroporation, insertion of a plasmid encasedin liposomes, or virus vectors may be used. Eukaryotic cells can also becotransformed with DNA sequences encoding the EspA of the invention, anda second foreign DNA molecule encoding a selectable phenotype, such asthe herpes simplex thymidine kinase gene. Another method is to use aeukaryotic viral vector, such as simian virus 40 (SV40) or bovinepapilloma virus, to transiently infect or transform eukaryotic cells andexpress the protein. (see for example, Eukaryotic Viral Vectors, ColdSpring Harbor Laboratory, Gluzinan ed., 1982).

[0054] Isolation and purification of microbial expressed polypeptide, orfragments thereof, provided by the invention, may be carried out byconventional means including preparative chromatography andimmunological separations involving monoclonal or polyclonal antibodies.

[0055] Among the prokaryotic organisms which may serve as host cells areE. coli strain JM101, E. coli K12 strain 294 (ATCC number 31,446), E.coli strain W3110 (ATCC number 27,325), E. coli X1776 (ATCC number31,537), E. coli XL-1Blue (Stratagene), and E. coli B; however, manyother strains of E. coli, such as HB101, NM522, NM538, NM539 and manyother species and genera of prokaryotes can be used as well. Besides theE. coli strains listed above, bacilli such as Bacillus subtillis, otherenterobacteriaceae such as Salmonella typhimunium or Serratia marcesansand various Pseudomonas species can all be used as hosts. In onespecific embodiment, the prokaryotic host cell is enteropathogenic E.coli. In another specific embodiment, the prokaryotic host cell israbbit enteropathogenic E. coli.

[0056] Among the eukaryotic organisms which may serve as host cells areyeast strains such as PS23-6A, W301-18A, LL20, D234-3, INVSC1, INVSC2,YJJ337. Promoter and enhancer sequences such as gal 1 and pEFT-1 areuseful. Vra-4 also provides a suitable enhancer sequence. Sequencesuseful as functional origins of replication include ars1 and 2μ circularplasmid.

[0057] The Gram-negative bacteria are a diverse group of organisms andinclude Spirochaetes such as Treponema and Borrelia, Gram-negativebacilli including the Pseudomonadaceae, Legionellaceae,Enterobacteriaceae, Vibrionaceae, Pasteurellaceae, Gram-negative coccisuch as Neisseriaceae, anaerobic Bacteroides, and other Gram-negativebacteria including Rickettsia, Chlamydia, and Mycoplasma.

[0058] Gram-negative bacilli (rods) are important in clinical medicine.They include (1) the Enterobacteriaceae, a family which comprises manyimportant pathogenic genera, (2) Vibrio, Campylobacter and Helicobactergenera, (3) opportunistic organisms (e.g., Pseudomonas, Flavobacterium,and others) and (4) Haemophilus and Bordetella genera. The Gram-negativebacilli are the principal organisms found in infections of the abdominalviscera, peritoneum, and urinary tract, as well secondary invaders ofthe respiratory tracts, burned or traumatized skin, and sites ofdecreased host resistance. Currently, they are the most frequent causeof life-threatening bacteremia. Examples of pathogenic Gram-negativebacilli are E. coli (diarrhea, urinary tract infection, meningitis inthe newborn), Shigella species (dysentery), Salmonella typhi (typhoidfever), Salmonella typhimurium (gastroenteritis), Yersiniaenterocolitica (enterocolitis), Yersinia pestis (black plague), Vibriocholerae (cholera), Campylobacter jejuni (enterocolitis), Helicobacterjejuni (gastritis, peptic ulcer), Pseudomonas aeruginosa (opportunisticinfections including burns, urinary tract, respiratory tract, woundinfections, and primary infections of the skin, eye and ear),Haemophilus influenzae (meningitis in children, epiglottitis, otitismedia, sinusitis, and bronchitis), and Bordetella pertussis (whoopingcough). Vibrio is a genus of motile, Gram-negative rod-shaped bacteria(family Vibrionaceae). Vibrio cholerae causes cholera in humans; otherspecies of Vibrio cause animal diseases. E. coli colonize the intestinesof humans and warm blooded animals, where they are part of the commensalflora, but there are types of E. coli that cause human and animalintestinal diseases. They include the enteroaggregative E. coli(EaggEC), enterohaemorrhagic E. coli (EHEC), enteroinvasive E.coli(EIEC), enteropathogenic E. coli (EPEC) and enterotoxigenic E. coli(ETEC). Uropathogenic E. coli (UPEC) cause urinary tract infections.There are also neonatal meningitis E. coli (NMEC). Apart from causingsimilar infections in animals as some of the human ones, there arespecific animal diseases including: calf septicaemia, bovine mastitis,porcine oedema disease, and air sac disease in poultry.

[0059] The Neisseria species include N. cinerea, N. gonorrhoeae, N.gonorrhoeae subsp. kochii, N. lactamica, N. meningitidis, N.polysaccharea, N. mucosa, N. sicca, N. subflava, the asaccharolyticspecies N. flavescens, N. caviae, N. cuniculi and N. ovis. The strainsof Moraxella (Branhamella) catarrhalis are also considered by sometaxonomists to be Neisseria. Other related species include Kingella,Eikenella, Sinonsiella, Alysiella, CDC group EF-4, and CDC group M-5.Veillonella are Gram-negative cocci that are the anaerobic counterpartof Neisseria. These non-motile diplococci are part of the normal floraof the mouth.

[0060] The pathogenic bacteria in the Gram-negative aerobic cocci groupinclude Neisseria, Moraxella (Branhamella), and the Acinetobacter. Thegenus Neisseria includes two important human pathogens, Neisseriagonorrhoeae (urethritis, cervicitis, salpingitis, proctitis,pharyngitis, conjunctivitis, pharyngitis, pelvic inflammatory disease,arthritis, disseminated disease) and Neisseria meningitides(meningitis,septicemia, pneumonia, arthritis, urethritis). Other Gram-negativeaerobic cocci that were previously considered harmless include Moraxella(Branhamella) catarrhalis (bronchitis and bronchopneumonia in patientswith chronic pulmonary disease, sinusitis, otitis media) has recentlybeen shown to be an common cause of human infections.

[0061] The EspA polypeptides of the invention can also be used toproduce antibodies which are immunoreactive or bind to epitopes of theEspA polypeptides. Antibody which consists essentially of pooledmonoclonal antibodies with different epitopic specificities, as well asdistinct monoclonal antibody preparations are provided. Monoclonalantibodies are made from antigen containing fragments of the protein bymethods well known in the art (Kohler, et al., Nature, 256:495, 1975;Current Protocols in Molecular Biology, Ausubel, et al., ed., 1989).

[0062] The term “antibody” as used in this invention includes intactmolecules as well as fragments thereof, such as Fab, Fab′, F(ab′)₂, andFv that can bind the epitope. These antibody fragments retain someability selectively to bind with its antigen or receptor and are definedas follows:

[0063] (1) Fab, the fragment that contains a monovalent antigen-bindingfragment of an antibody molecule can be produced by digestion of wholeantibody with the enzyme papain to yield an intact light chain and partof one heavy chain;

[0064] (2) Fab′, the fragment of an antibody molecule can be obtained bytreating whole antibody with pepsin, followed by reduction, to yield anintact light chain and part of the heavy chain; two Fab′ fragments areobtained per antibody molecule;

[0065] (3) (Fab′)₂, the fragment of the antibody that can be obtained bytreating whole antibody with the enzyme pepsin without subsequentreduction; F(ab′)₂ is a dimer of two Fab′ fragments held together by twodisulfide bonds;

[0066] (4) Fv, defined as a genetically engineered fragment containingthe variable region of the light chain and the variable region of theheavy chain expressed as two chains; and

[0067] (5) Single chain antibody, defined as a genetically engineeredmolecule containing the variable region of the light chain, the variableregion of the heavy chain, linked by a suitable peptide linker as agenetically fused single chain molecule.

[0068] Methods of making these fragments are known in the art. (See, forexample, Harlow and Lane, Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory, New York (current edition), incorporated herein byreference).

[0069] An epitope is any antigenic determinant on an antigen to whichthe paratope of an antibody binds. Epitopes usually consist ofchemically active surface groupings of molecules such as amino acids orsugar side chains and usually have specific three dimensional structuralcharacteristics, as well as specific charge characteristics.

[0070] If needed, polyclonal or monoclonal antibodies can be furtherpurified, for example, by binding to and elution from a matrix to whichthe peptide or a peptide to which the antibodies are raised is bound.Those of skill in the art will know of various techniques common in theimmunology arts for purification and/or concentration of polyclonalantibodies, as well as monoclonal antibodies (See, e.g., Coligan, etal., Unit 9, Current Protocols in Immunology, Wiley Interscience,current edition, incorporated by reference).

[0071] The invention also provides peptide epitopes for use in designingespA specific nucleotide probes or anti-EspA antibodies. Such probes orantibodies can be used to identify proteins or genes that may beinvolved in the virulence of other pathogens, including but not limitedto polypeptides or polynucleotides from Gram-negative bacteria.

[0072] The antibodies of the invention, including polyclonal andmonoclonal antibodies, chimeric antibodies, single chain antibodies andthe like, have with the ability to bind with high immunospecificity tothe EspA proteins, peptides or nucleotide sequences of the invention, orfragments thereof. These antibodies can be unlabeled or suitablylabeled. Antibodies of the invention can be used for affinitypurification of EspA for example. Antibodies of the invention may beemployed in known immunological procedures for qualitative orquantitative detection of these proteins or peptides in cells, tissuesamples, sample preparations or fluids. Antibodies of the invention maybe employed in known immunological procedures for qualitative orquantitative detection of the nucleotide sequences or portions thereof.

[0073] The invention provides a method for detecting EspA polypeptide ina sample, including contacting a sample from a subject with an antibodyto EspA polypeptide; and detecting binding of the antibody to EspApolypeptide. Binding is indicative of the presence of EspA polypeptidein the sample. As used herein, the term “Sample” includes materialderived from a mammalian or human subject or other animal. Such samplesinclude but are not limited to hair, skin samples, tissue samplecultured cells, cultured cell media, and biological fluids. For example,EspA polypeptide can be detected in HeLa cell (e.g., human) culture.

[0074] As used herein, the term “tissue” refers to a mass of connectedcells (e.g., CNS tissue, neural tissue, or eye tissue) derived from ahuman or other animal and includes the connecting material and theliquid material in association with the cells. For example, rabbitenteropathogenic E. coli can be found in the stomach, cecum and colon ofrabbits. As used herein, the term “biological fluid” refers to liquidmaterial derived from a human or other animal. Such biological fluidsinclude but are not limited to blood, plasma, serum, serum derivatives,bile, phlegm, saliva, sweat, antiotic fluid, and cerebrospinal fluid(CSF), such as lumbar or ventricular CSF.

[0075] As used herein, the term “sample” also includes solutionscontaining the isolated polypeptide, media into which the polypeptidehas been secreted, and media containing host cells which produce theEspA polypeptide. For example, a sample may be a protein samples whichis to be resolved by SD S-PAGE and transferred to nitrocellulose forWestern immunoblot analysis. The quantity of sample required to obtain areaction may be determined by one skilled in the art by standardlaboratory techniques. The optimal quantity of sample may be determinedby serial dilution.

[0076] In one embodiment, the presence of EspA polypeptide in the sampleis indicative of infection by enteropathogenic E. coli. In anotherembodiment, the presence of EspA polypeptide in the sample is indicativeof infection by enterohemorrhagic E. coli.

[0077] Proteins, protein fragments, and synthetic peptides of theinvention are projected to have numerous uses including prognostic,therapeutic, diagnostic or drug design applications. Proteins, proteinfragments, and synthetic peptides of the invention will provide thebasis for preparation of monoclonal and polyclonal antibodiesspecifically immunoreactive with the proteins of the invention. In oneembodiment, the invention provides a method of immunizing a hostsusceptible to disease caused by EspA-producing E. coli, byadministering to a host with the polypeptide of claim 1; and inducing aprotective immune response in the host to EspA polypeptide. Theinfection of the host by EspA-producing organism is thereby prevented.In a more specific embodiment, the EspA-producing organism is an E. colistrain. In an even more specific embodiment, the E. coli strain iseither enteropathogenic or enterohemorrhagic E. coli.

[0078] In another embodiment, the invention provides a method ofameliorating disease caused by EspA-producing organism, by immunizing ahost with EspA polypeptide and inducing an immune response in the hostto the EspA polypeptide. In a more specific embodiment, theEspA-producing organism is an E. coli strain. In an even more specificembodiment, the E. coli strain is either enteropathogenic orenterohemorrhagic E. coli. The invention provides a method for detectingespA polynucleotide in a sample, by contacting a sample suspected ofcontaining espA polynucleotide with a nucleic acid probe that hybridizesto espA polynucleotide; and detecting hybridization of the probe withespA polynucleotide. The detection of hybridization is indicative ofespA polynucleotide in the sample.

[0079] In another embodiment, the invention provides an organism with amutated espA gene. Preferred organisms in which an espA gene may bemutated include but are not limited to bacteria. Among the bacteria inwhich an espA gene may be mutated are E. coli. Among the E. coli inwhich an espA gene may be mutated are enteropathogenic andenterohemorrhagic E. coli.

[0080] The invention provides a recombinant method for producing espApolynucleotide, including inserting a nucleic acid encoding a selectablemarker into the polynucleotide encoding EspA polypeptide. The resultingpolynucleotide encodes a recombinant EspA polypeptide containing theselectable marker. For example, a selectable marker may be a herpessimplex virus (HSV) tag, for which there are commercially availableantibodies.

[0081] The invention provides a recombinant method for producing EspApolypeptide, by growing a host cell containing a polynucleotide encodingEspA polypeptide under conditions which allow expression and secretionof EspA polypeptide; and isolating the polypeptide. Methods of producingpolypeptides and peptides recombinantly are within the scope of thisinvention. As used herein, the term “conditions which allow expressionand secretion” refers to suitable conditions such that the nucleic acidis transcribed and translated and isolating the polypeptide so produced.The polypeptide produced may be a protein secreted into the media. Mediaincludes a fluid, substance or organism where microbial growth can occuror where microbes can exist. Such environments can be, for example,animal tissue or bodily fluids, water and other liquids, food, foodproducts or food extracts, and certain inanimate objects. For example,microbes may grow in Luria-Bertani (LB) media. It is not necessary thatthe environment promote the growth of the microbe, only that it permitsits subsistence.

[0082] The invention provides a method to identify a compound whichinhibits bacterial type III secretion systems, by introducing thepolynucleotide encoding a selectable marker into bacteria having abacterial type III secretion system; growing the bacteria underconditions which allow growth of bacteria and secretion of thepolypeptide encoded by the polynucleotide; contacting a compoundsuspected of inhibiting the bacterial type III secretion system with thebacteria; inducing the expression of the polypeptide; and detecting thesecretion of the polypeptide. In the practice of the method, a lack ofsecretion is indicative of the inhibition of bacterial type IIIsecretion systems. As used in this invention, the term “type IIIsecretion” and “type III secretion” pathway refer to a specializedmachinery to export molecules across a cell membrane. Exportingmolecules across a cell membrane is a process critical for movingvirulence factors to the surface where they can interact with host cellcomponents. The type III secretion pathway uses adenosine triphosphate(ATP) as an energy source. The type III secretion pathway is differentthan other secretion pathways found in Gram-negative bacteria, althoughit is homologous to flagella and filamentous phage assembly genes. Itdoes not resemble any mammalian pathway. It is always associated withdisease production. The virulence factors secreted by the type IIIsecretion pathway vary between pathogens, although components of thetype III secretion machinery are interchangeable, at least forSalmonella, Shigella, and Yersinia.

[0083] Furthermore, the polypeptide or nucleotide sequences of theinvention can be used to identify compounds or compositions whichinteract (e.g., bind) with them and affect their biological activity.Such effects include inhibition or stimulation of EspA activity orsecretion.

[0084] The invention provides a method for producing a nonpathogenicorganism, by generating a mutation in a polynucleotide encoding EspApolypeptide; inserting a nucleic acid sequence encoding a selectablemarker into the site of the mutation; introducing the mutated espApolynucleotide into a chromosomal espA gene of an organism to produce amutation in the chromosomal espA gene; and selecting organisms havingthe mutation. As used herein, the term “mutation” refers to a change inthe nucleotide sequence of a gene, in particular, the polynucleotideencoding EspA polypeptide. Mutations include mutations producing EspApolypeptide with a different amino acid sequence, missense mutations(including frame shift mutations), nonsense mutations (includingknockout mutations), and recombinant genetic techniques which producefusion proteins containing part of the EspA polypeptide. In oneembodiment, the nucleic acid sequence encoding a selectable markerencodes resistance to kanamycin. For example, the aphA-3 cassette,encoding a gene for resistance to kanamycin (kan), may be cloned intothe polynucleotides encoding the EspA polypeptide for selection of themutated espA polynucleotide on kanamycin plates to produce a knockoutmutation.

[0085] Preferred organisms in which to practice the invention includebut are not limited to bacteria. In another embodiment, the organismwhich is used to generate a mutation in a polynucleotide encoding EspApolypeptide is E. coli. Among the E. coli that may be transformed areenteropathogenic and enterohemorrhagic E. coli.

[0086] The invention provides a method of activating tyrosine kinaseactivity in a host cell by adding both mutant espA-deficient organismsthat express Eae polypeptide and mutant eaeA-deficient organisms thatexpress EspA polypeptide to a host cell and binding the bacteria to thehost cell, thereby activating host cell tyrosine kinase activity in thecell. In one embodiment, the activation of host cell tyrosine kinaseactivity in the cell causes the tyrosine phosphorylation of a 90kilodalton host membrane protein, Hp90, and fluxes of intracellularinositol phosphate (IP₃) and calcium. For example, an eaeA mutant can beused to complement an espA mutant for invasion when these two mutantstrains were used to co-infect HeLa cells. The invention thus provides auseful scientific method to investigate pathogenesis by cell biology.

[0087] This invention includes a kit containing one or more antibodiesof the invention as well as a nucleotide based kit. In one embodiment,the kit is useful for the detection of EspA polypeptide and is a carriermeans compartmentalized to receive in close confinement a containercontaining an antibody which binds to EspA polypeptide. As used herein,a “container means” includes vials, tubes, and the like, each of thecontainer means comprising one of the separate elements to be used inthe method. In one embodiment, the antibody which binds to EspApolypeptide is detectably labeled. In a more specific embodiment, thelabel is selected from the group consisting of radioisotope, abioluminescent compound, a chemiluminescent compound, a fluorescentcompound, a metal chelate, and an enzyme.

[0088] In another embodiment, the kit is useful for the detection of anespA polynucleotide and is a carrier means compartmentalized to receivein close confinement a container containing the nucleic acid probe thathybridizes to espA polynucleotide. In one embodiment, nucleic acid probethat hybridizes to espA polynucleotide is detectably labeled. In a morespecific embodiment, the label is selected from the group consisting ofradioisotope, a bioluminescent compound, a chemiluminescent compound, afluorescent compound, a metal chelate, and an enzyme.

[0089] Since EspA is a secreted protein, it is useful as a fusionpartner for cloning and expressing other peptides and proteins. Forexample, EspA fused to a protein of interest is recombinantly producedin a host cell, e.g., E. coli, and the fusion protein is secreted intothe culture media in which the transformed host is grown. The fusionprotein can be isolated by anti-EspA antibodies followed by cleavage ofEspA from the peptide or protein of interest. ELISA or otherimmunoaffinity methods can be used to identify the EspA fusion protein.The invention provides a method of producing an EspA fusion proteinincluding growing a host cell containing a polynucleotide encoding EspAoperably linked to a polynucleotide encoding a polypeptide or peptide ofinterest under conditions which allow expression and secretion of thefusion polypeptides and isolating the fusion polypeptide. The term“operably linked or associated” refers to functional linkage between apromoter sequence and the structural gene or genes in the case of afusion protein, regulated by the promoter nucleic acid sequence. Theoperably linked promoter controls the expression of the polypeptideencoded by the structural gene (e.g., the fusion protein).

[0090] The following examples are intended to illustrate but not limitthe invention. While they are typical of those that might be used, otherprocedures known to those skilled in the art may alternatively beutilized.

EXAMPLE 1 DNA Sequence Analysis of the Enteropathogenic E. coli espAGene

[0091] The purpose of this Example is to characterize espA, a generesponsible for the attaching and effacing activity of enteropathogenicE. coli. The espA gene encodes the 25 kilodalton secreted protein and islocated on the enteropathogenic E. coli genome in the Locus ofEnterocyte Effacement between eaeA and espB, two loci required forintimate adherence.

[0092] The DNA sequence of the enteropathogenic E. coli Locus ofEnterocyte Effacement between eaeA and espB was determined. DNAsequencing was performed as follows: The SalI-BglII DNA fragment of theLocus of Enterocyte Effacement spanning from within eaeA to upstream(5′) of espB was cloned into the commercially available plasmidpBluescript to create the plasmid pLCL109. A series of nested DNAdeletions was made from the end of the plasmid pLCL109 closer to theeaeB gene. These DNA deletions of plasmid pLCL109 were used as templatesto determine the nucleotide sequence of both strands of DNA usingoligonucleotide primers synthesized as needed, [a-³⁵S]dATP, and theSequenase enzyme. DNA sequence data were analyzed with the packagedeveloped by the Genetics Computer Group of the University of Wisconsin.

[0093] Analysis of the DNA sequence showed three open reading frames.The amino acid sequence predicted by the DNA sequence at the 5′ end ofthe second open reading frame (espA) was identical to the amino-terminalsequence of a protein with an M_(R) of approximately 25 dalton secretedby enteropathogenic E. coli. No proteins with similar sequences weredetected in a search of the Genbank, database using the TFASTA.Therefore the espA gene encodes the secreted enteropathogenic E. coliprotein.

[0094] The predicted molecular weight of the entire protein encoded byespA is 20,468 daltons (FIG. 1). No leader sequence precedes theamino-terminus of the reported secreted product. A strong consensusribosome binding site ends seven nucleotides prior to the start codon.Eleven of the first nineteen residues at the amino-terminus werepredicted to be serine or threonine.

EXAMPLE 2 Construction of a Mutation in an espA Gene on a Plasmid and ona Chromosome

[0095] The purpose of this Example was to construct a mutation in anespA gene on the chromosome of an organism. The purpose of this Examplewas to construct a mutation in an espA gene on a plasmid. The plasmidcan then be used to create mutations in the espA gene of otherorganisms. Plasmids with a nonpolar mutation in espA gene wereconstructed. Furthermore, a mutant bacterial strain with a nonpolarmutation in its chromosomal espA gene was generated.

[0096] An espA gene with a nonpolar mutation is described in FIG. 2. Theuse of the polymerase chain reaction (PCR) generated a deletion withrestriction sites that allow an aphA-3 cassette, encoding resistance tokanamycin, to be cloned into the deleted region. This aphA-3 cassette ispreceded (5′) by translation stop codons in all three reading frames andimmediately followed (3′) by a consensus ribosome binding site and astart codon. The insertion into the espA gene was engineered to retainthe reading frame of the 3′ end of espA and to therefore allowunaffected transcription and translation of downstream (3′) genes. DNAsequencing confirmed the reading frame of the mutation.

[0097] The construction of a nonpolar mutation in the espA gene on aplasmid was performed by the polymerase chain reaction as follows: ThePCR template was plasmid pLCL114, containing the ClaI-BglII fragment ofpLCL109 cloned into pBluescript. Two pairs of primers were used, theuniversal primer with Donne-99 and the reverse primer with Donne-100.Oligonucleotides Donne-99 and Donne-100 are nucleotides 4157 through4140 and 4297 through 4324 of the SalI-BglII fragment, respectively. Forcloning purposes, an NruI restriction site was engineered into the 5′end of both Donne-99 and Donne-100. Oligonucleotides were constructed atthe Biopolymer Laboratory of the University of Maryland at Baltimore.PCR was performed on 50 μL samples in a minicyler. The PCR reaction wasthirty cycles of DNA denaturation at 94° C. for one minute, annealing tothe primers at 55° C. for two minutes, and polynucleotide extension at72° C. for three minutes. The two resulting amplified fragmentsamplified were each cloned into the commercially available plasmidpCRscript to create pLCL119 and pLCL120, respectively. The insert ofpLCL120 was then cloned into pLCL119 using SalI and NruI to createpLCL21 containing the desired deletion. The 850 base pair aphA-3kanamycin resistance cassette flanked by SmaI sites was then insertedinto the NruI site of pLCL121.

[0098] The mutated espA allele was cloned in the positive-selectionsuicide vector pCVD442 and introduced into wild-type enteropathogenic E.coli strain E2348/69 by allelic exchange. A espA mutant bacterial strainwas constructed as follows: The SalI-SacI fragment from the plasmid withthe aphA-3 kanamycin resistance which contained the interrupted espAgene was cloned into positive-selection suicide vector pCVD442 inDH5αpir for introduction into E2348/69 by triparental conjugation or byelectroporation in 0.1 cm cuvettes with an E. coli pulser set at 1.8 kV.

[0099] An espA mutant was selected on modified LB kanamycin plates. Theresulting enteropathogenic E. coli mutant strain, UMD872, was resistantto sucrose and kanamycin, and sensitive to ampicillin. PCR amplificationusing the two primers flanking the mutation, Donne-52 and Donne-73,confirmed the construction of the espA mutation. Bacteria were stored at−70° C. in 50% LB broth/50% glycerol (vol/vol) and grown on LB agarplates or LB broth with chloramphenicol (20 μg/ml), ampicillin (200μg/ml), nalidixic acid (50 μg/ml), or kanamycin (50 μg/ml) added asneeded.

EXAMPLE 3 Disruption of the enteropathogenic E. coli espA Gene AbolishesSecretion of the EspA Protein.

[0100] The purpose of this Example was to identify the protein encodedby the espA gene. A comparison of the amino-terminal sequence data ofthe EspA protein agreed with the translatedespA gene sequence. Toconfirm this result, the espA deletion mutant, UMD872, was grown inradiolabeled tissue culture media. The deletion of the espA gene resultsin the loss of the 25 kilodalton radiolabeled secreted protein. Thisresult was confirmed by immunoprecipitation using an anti-EPEC antiserawhich reacts with the secreted EspA protein. Western analysis usinganti-EPEC antisera showed no 25 kilodalton secreted protein. Secretionof the EspA protein was restored when the espA deficient strain wastransformed a plasmid (pMSD2) with an intact espA. The increasedproduction by the bacteria of EspA protein encoded by the plasmidreduced the secretion of the other proteins by the type III secretionpathway.

EXAMPLE 4 Enteropathogenic E. coli EspA is Required for Invasion ofCultured Epithelial Cells

[0101] The purpose of this Example was to examine whether theenteropathogenic E. coli EspA protein is involved in epithelial cellinvasion. EspA protein is needed for triggering the host signaltransduction pathway and invasion of host cells.

[0102] Monolayers of an epithelial cancer cell (HeLa) were infected forthree hours with either parental wild-type or espA mutantenteropathogenic E. coli strains. The number of adherent andintracellular (i.e., invasive) enteropathogenic E. coli was determined.The absolute number of bacteria adherent to HeLa cells varied betweenstrains, according to the different growth rates between the mutant andparental enteropathogenic E. coli strains. While the espA mutant strainUMD872 adheres efficiently to epithelial cells, it is deficient forinvasion. However, UMD872 invaded HeLa monolayers at near wild-typelevels when the espA gene was genetically complemented by the plasmidpMSD2, which encodes an intact espA gene.

[0103] Co-infection experiments of HeLa monolayers were carried out todetermine whether the invasion defective behavior of bacterial strainswith mutations in either espA, espB, and eaeA could geneticallycomplement each other in trans to mediate subsequent invasion. The eaeAbacterial mutant strain activates signaling but lacks intimateadherence. Signaling mediated by an eaeA mutant strain allows an espBdeficient strain to enter epithelial cells, but not the converse. TheeaeA mutant complemented an espA mutant for invasion when these twomutant strains were used to co-infect HeLa cells, but there was noincrease in invasion of the eaeA mutant strain. Indeed, the signalinginduced by the more adherent eaeA mutant lead to the increased uptake ofthe espA mutant. Co-infection with espA and espB deleted strains did notenhance invasion of either strain, showing that espA and espB do notcomplement each other.

[0104] Co-infection experiments demonstrated that like espB, the espAmutant behavior was complemented by eaeA but not the reverse. Indeed,the signaling generated by the more adherent eaeA mutant lead to theincreased uptake of the intimin expressing espA mutant. In contrast,neither the espA nor the espB mutant strain could complement each other,implying that both proteins may act together at the same step to induceepithelial signaling.

EXAMPLE 5 EspA is Essential to Induce Signal Transduction Events inEpithelial Cells

[0105] The purpose of this Example was to determine whether EspA isessential to induce signal transduction events in epithelial cells.Enteropathogenic E. coli induce tyrosine phosphorylation of a host cell90 kilodalton membrane protein and subsequent accumulation ofphosphorylated proteins, actin, and other cytoskeletal componentsbeneath adherent bacteria. The ability of an espA mutant defective forinvasion to induce these two signaling events in mammalian cells wasexamined. Unlike wild-type enteropathogenic E. coli, the espA mutantstrain UMD872 was unable to induce phosphorylation of host Hp90. Theability to induce this phosphorylation event was restored by a plasmid(pMSD2) that encodes the EspA protein.

[0106] The adherence and invasion assays were performed as follows: 10⁵HeLa cells grown in DMEM were infected with the various enteropathogenicE. coli strains (m.o.i. 1:100) for three hours. HeLa cells were grown at37° C. with 5% CO₂ in Dulbecco's Minimal Eagles Medium (DMEM)supplemented with 10% (vol/vol) fetal calf serum. Monolayers were washedthrice in phosphate-buffered saline before lysing in 1% Triton (vol/vol)in phosphate-buffered saline and plating out serial dilution on LB agarplates. For invasion assays the washed monolayers were incubated withgentamicin (100 μg/ml) for one hour to kill external bacteria beforewashing, lysing and plating out.

[0107] HeLa cell monolayers were infected for three hours with wild-typeor mutant enteropathogenic E. coli strains. The epithelial Triton X-100soluble and insoluble proteins of HeLa cells were isolated. Proteinsamples were resolved by SDS-PAGE and transferred to nitrocelluloseprior to probing with anti-phosphotyrosine specific antibodies.

[0108] The isolation of enteropathogenic E. coli-secreted proteins andHeLa cellular proteins was accomplished as follows: Tissue cultureplates were seeded overnight with 10⁶ HeLa cells. Before infection, themedia was replaced with DMEM minus methionine/cysteine containingcycloheximide (100 μg/ml). HeLa cells were grown at 37° C. with 5% CO₂in DMEM supplemented with 10% (vol/vol) fetal calf serum.Enteropathogenic E. coli was added (m.o.i. 100:1) and incubated for 2.5hours at 37° C. in 5% CO₂ incubator before adding 200 μg/ml ³⁵Scysteine/methionine for 30 minutes. The culture supernatant was removedand the bacteria pelleted by centrifugation (18,000× g, 10 minutes). Thesupernatant secreted proteins were precipitated by the addition of icecold trichloroacetic acid (10% vol/vol) and incubated on ice for 60minutes. Proteins were pelleted by centrifugation as above andresuspended in Laemelli sample buffer. Samples were resolved by 12%SDS-PAGE and protein profiles examined by autoradiography or transferredto nitrocellulose prior to probing with anti-EPEC antibodies.

[0109] All enteropathogenic E. coli strains exhibited a tyrosinephosphorylated 85 kilodalton protein Ep85 in the insoluble fraction,confirming the presence of the enteropathogenic E. coli strains on themonolayer.

[0110] HeLa phosphotyrosine proteins were analyzed as follows: InfectedHeLa monolayers were washed thrice with ice cold phosphate-bufferedsaline prior to lysis in 1% Triton X-100 in the presence of proteaseinhibitors. The Triton insoluble and soluble fraction were isolated,resuspended in Laemelli sample buffer, and analyzed for the presence ofphosphotyrosine proteins by Western immunoblot analysis withanti-phosphotyrosine antibodies.

[0111] Examination of infected HeLa cells by immunofluorescencemicroscopy with fluorescently labeled anti-phosphotyrosine antibodies orrhodamine-phalloidin showed that, unlike wild-type parentalenteropathogenic E. coli, the espA mutant did not accumulate tyrosinephosphorylated proteins or cytoskeletal actin beneath the adherentmicrocolonies. However, phosphotyrosine and actin accumulation could berestored by using a strain carrying the espA gene on the plasmid pMSD2.

[0112] Immunofluorescence microscopy was performed as follows: HeLacells which were seeded on round glass cover slips were infected withenteropathogenic E. coli or a mutant strain for three hours. Themonolayers were then washed and fixed in 2.5% paraformaldehyde prior tostaining for filamentous actin (using phallodin-rhodamine) or withanti-phosphotyrosine antibodies with an appropriate secondaryfluorescein conjugated antibody.

EXAMPLE 6 Characterization of Rabbit Enteropathogenic E. coli (RDEC-1)Secreted Virulence Proteins, EspA and EspB

[0113] The purpose of this Example was to investigate the structure ofEspA and EspB in rabbit enteropathogenic E. coli (RDEC-1). The espA andespA genes were cloned and their sequences were compared to those ofenteropathogenic E. coli (EPEC). The EspA protein showed some similarity(88.5% identity). The EspB protein was heterogeneous in internal regions(69.8% identity), but was identical to one strain of enterohemorrhagicE. coli (EHEC).

[0114] Cloning and sequence analysis of espA and espB genes was done asfollows: The DNA fragment encoding RDEC-1 espA and espB was obtained byPCR from RDEC-1 chromosomal DNA using primers derived from the publishedsequence of enteropathogenic E. coli. Vent DNA polymerase was used forPCR to amplify chromosomal DNA from RDEC-1 and enteropathogenic E. colistrains. The PCR reaction was carried out for thirty cycles ofdenaturation at 94° C. for one minute, annealing at 55° C. for oneminute, and elongation at 72° C. for two minutes. The resulting 4.3kilobase pair product was ligated into the commercially availableplasmid pBluescript and both strands were sequenced. DNA sequencing wasdone as follows: The 4.3 kilobase pair DNA fragment encoding the espAand espB genes was amplified by PCR using the primers AA01(+) andMS11(−), and RDEC-1 chromosomal DNA as the DNA template. The resultingblunt end fragment was digested with SalI and cloned into the SalI-SmaIsite of the commercially available plasmid pBluescript-II SK (+). TheDNA sequence of RDEC-1 espA and both strands using the commerciallyavailable Taq DyeDeoxy kit. (FIG. 3)

[0115] Two open reading frames were found in the cloned region and theseDNA sequences were similar to enteropathogenic E. coli espA and espB.The predicted molecular weight of RDEC-1 EspA (192 amino acids) was23,533 dalton, and RDEC-1 EspB (314 amino acids) was 33,219 dalton.RDEC-1 EspA was somewhat similar to that of enteropathogenic E. coliwith 88.5% identity (FIGS. 4A and 4B).

[0116] In an unexpected result, RDEC-1 EspB protein was identical to therecently reported EspB from enterohemorrhagic E. coli strain 413/89-1serotype 026, which was originally isolated from a calf and alsoisolated from patients with hemolytic uraemic syndrome, although twonucleotide differences occurred at positions 12 (T to C) and 729 basepair (G to T). Furthermore, RDEC-1 EspB showed 70.3% enc. 69.8% identityrespectively to that of enterohemorrhagic E. coli serotype 0157 andenteropathogenic E. coli strains. Small sequence deletions were found inRDEC-1 and enterohemorrhagic E. coli (serotype 026 and 0157) EspB at thesame positions when compared to the enteropathogenic E. coli sequences(FIGS. 4A-C).

[0117] These results show that RDEC-1 encodes espA and espB genes, andthat the predicted EspA polypeptide is highly conserved in RDEC-1 andenteropathogenic E. coli. However EspB is more similar to that ofenterohemorrhagic E. coli rather than enteropathogenic E. coli. An openreading frame downstream (3′) from espA showed similarity toenteropathogenic E. coli EspD, a secreted protein that modulates EspBsecretion and is needed for triggering of host signal transductionpathways (EMBL GenBank data, Accession No. Y09228). These results showthat espD is also located between espA and espB in RDEC-1.

EXAMPLE 7 Characterization of RDEC-1 EspA and EspB

[0118] The purpose of this Example was to investigate the function ofEspA and EspB in rabbit enteropathogenic E. coli (RDEC-1). Mutations inRDEC-1 espA and espB revealed that the RDEC-1 gene products areessential for triggering of host signal transduction pathways andinvasion into HeLa cells. Complementation with plasmids containingenteropathogenic E. coli espA and espB into RDEC-1 mutant strainsdemonstrated that they were functionally interchangeable, although theenteropathogenic E. coli proteins mediated higher levels of invasion.Furthermore, maximal expression of RDEC-1 and enteropathogenic E. colisecreted proteins occurred at their respective host's body temperatures,which may contribute to lack of enteropathogenic E. coli infectivity inrabbits.

[0119] To confirm the role of RDEC-1 espA and espB in host epithelialsignal transduction pathways, a non-polar stop codon mutation wasengineered into espA and espB. Two suicide vectors were constructed andintroduced into the RDEC-1 wild type strain by back conjugation. Theresulting mutant strains, AAF001ΔA (EspA⁻), AAF001ΔB (EspB⁻), and doublemutant strain AAF001ΔAB (EspA⁻/EspB⁻) were confirmed by BglII digestion.

[0120] The construction of the non-polar stop codon mutations in RDEC-1espA and espB genes was performed as follows: The 2.7 kilobase pair DNAfragment of the Locus of Enterocyte Effacement encoding the espA andespB genes was amplified by PCR using the primers BK25(+) and MSll(−),and pORF123B as the DNA template. The resulting blunt end fragment wasdigested with EcoRI and cloned into the EcoRI-SmaI site of pBluescriptII SK(+) vector to obtain pORF23B.A 1.1 kilobase pair EcoRI-BglII DNAfragment from pORF23B containing espA was cloned into the EcoRI-BamHIsite of pBluescript II SK(+) to obtain pORF23.

[0121] To construct a non-polar mutation in espA, inverse PCR wascarried out using the ΔespA(+) and ΔespA(−) primers which contain aBglII restriction site and a stop codon using circular pORF23 as a DNAtemplate. The PCR product was then blunt end ligated to obtain pORF23Δ.The resulting plasmid contained a stop codon and a BglII site 235 basepair downstream (3′) from the espA start codon, which was confirmed byDNA sequencing. The 1.1 kilobase pair SalI-SacI DNA fragment containingthe espA mutation from pORF23A was inserted into the same sites of thesuicide vector pCVD442, which contains the sacB gene for positiveselection and an ampicillin resistance gene, to obtain pAA23Δ. Theresulting plasmid was introduced into E. coli SM10λpir and backconjugated into RDEC-1 harboring pACYC184.

[0122] For the non-polar mutation in espB, inverse PCR was carried outusing the ΔespB(+) and ΔespB(−) primers, and pBxb as a DNA template.pBxb contains the 1.4 kilobase pair XbaI fragment from pORF23B encodingespB cloned into the pBluescript vector. The resulting PCR product wasself-ligated to obtain pBxbΔ that contained a stop codon and a BglIIsite introduced by the ΔespB(−) and ΔespB(+) primers. The resulting espgene in pBXbΔ was deleted by 250 base pair, starting 154 base pairdownstream (3′) of the espB start codon. The 1.1 kilobase pair SalI-SacIsite DNA fragment containing the espB mutation from pBxbA was insertedinto the same site of the pCVD442 to obtain pAABxbΔ. The resultingplasmid was transformed into E. coli SM10λpir and back conjugated intoRDEC-1 harboring pACYC184. To establish double mutations in espA andespB, pAABxbΔ was introduced into AAF001ΔA (EspA⁻) strain. Three RDEC-1non-polar mutant strains were confirmed by their phenotypes whichmaintain resistance to sucrose and chloramphenicol, and sensitivity toampicillin. To confirm the stop codon insertions in espA and espB,chromosomal DNA was prepared from each mutant strain and PCR wasperformed with primers encompassing the esp genes. The resulting PCRproducts were digested with BglII to confirm the presence of thisengineered restriction site. The mutant strains containing the stopcodon in espA or/and espB were designated as AAF001ΔA (EspA⁻), AAF001ΔB(EspB⁻) and AAF001ΔAB (EspA⁻/EspB⁻), respectively.

[0123] Mutations back to wild type (“back mutation”) were made toconfirm that suicide vectors do not affect respective flanking region orother loci. Two back mutant strains were obtained by bans conjugation ofthe suicide vectors pAA23 and pAABxb into AAF001ΔA (EspA⁻) and AAF001ΔB(EspB⁻) strains. The resulting back mutant strains, AAF003 and AAF004,were confirmed by PCR and BglII digestion. The construction of backmutations in EspA and EspB strains was done as follows: The 1.1 kilobasepair SalI-SacI DNA fragment from pORF23 containing espA was insertedinto the SalI-SacI sites of pCVD442 to obtain pAA23. The 1.4 kilobasepair SalI-SacI fragment of pBxb was inserted into the SalI-SacI site ofpCDD442 to obtain pAAFBxb. pAA23 and pAABxb were introduced into SMλpirand bans conjugated into AAF001AA and AAF001AB. The resulting backmutant strains were confirmed as described above and designated asAAF002 (EspA+) and AAF003 (EspB+).

[0124] The cloning of the enteropathogenic E. coli espA and espB geneswas done as follows: The 2.8 kilobase pair DNA fragment encoding espAand espB was amplified by PCR using the primers EespA(+) and EespB(−)with enteropathogenic E. coli 2348/69 chromosomal DNA as the template.This fragment was digested with BamHI and SalI and introduced into theBamHI-SalI site of the low-copy vector pMW118 under control of the lacZpromoter, to obtain pMWespAB. pMWespAB was digested with BglII which hasa restriction site in espD open reading frame, blunt ended with Klenowfragment, and then self-ligated to obtain pMW6espD. pMWespAB was alsodigested with BglII and BamHI, and then self ligated to obtain pMWespB.The PMWespAB was digested with BglII-SalI, and filled with KlenowFragment, then self-ligated to obtain pMWespA.

[0125] The secretion profile of RDEC-1 and its mutant strains in tissueculture media was analyzed. Enteropathogenic E. coli secretes fiveproteins, 110 kilodalton (EspC), 40 kilodalton, 39 kilodalton, 37kilodalton (EspB), and 25 kilodalton (EspA) in culture media. RDEC-1showed a similar secretion profile, except it did not secrete a proteinequivalent to EspC. EspC is not required for enteropathogenic E. coliinduction of host signal transduction pathways. Although two secretedproteins (40 and 39 kilodalton) were difficult to resolve, theseproteins could be resolved using different conditions of SDS-PAGE.RDEC-1 secreted two proteins with similar mobility to enteropathogenicE. coli EspA and EspB.

[0126] RDEC-1 secreted proteins were prepared as follows: Bacterialovernight cultures were diluted 1:100 into DMEM and incubated to anoptical density of 1.0 at 600 an (OD600). For RDEC-1 mutant strainscontaining enteropathogenic E. coli espA and espB recombinant plasmids,isopropylthiogalactoside (IPTG) was added in DMEM to inducetranscription. Bacteria were removed by centrifugation (18,000× g, 10minutes) and the supernatant precipitated by addition of 10% ice-coldtrichloroacetic acid, and incubated on ice for one hour. Aftercentrifugation, the pellets were resuspended in Laemmli sample bufferand analyzed by 12% SDS-PAGE.

[0127] Both EspA and EspB proteins cross-react to anti-enteropathogenicE. coli EspA and anti-enteropathogenic E. coli EspB antisera in Westernimmunoblots, indicating that RDEC-1 also secretes EspA and EspBproteins. Rabbit polyclonal antibodies against enteropathogenic E. coliEspA and EspB were used in Western blots.

[0128] Mutant strains AAF001ΔA, AAF001ΔB, and AAF001ΔAB lack secretionof EspA, EspB, and EspA/EspB proteins, respectively, as judged by theirsecretion profile and Western blot analysis. EspB, whose gene is locateddownstream (3′) from espA, was still secreted in the mutant strainAAF00ΔA (EspA⁻), indicating that the stop codon insertion mutation doesnot affect downstream gene expression. These results also confirm thatRDEC-1 EspA and EspB proteins are encoded by the sequences we designatedas espA and espB. Furthermore, the two back mutant strains AAF002 andAAF003, that were originally derived from AAF001ΔA (EspA⁻) and AAF001ΔB(EspB⁻), now expressed the parental secreted proteins indicating thateach non-polar mutation in AAF001ΔA and AAF001ΔB is as predicted, anddoes not affect downstream genes and other loci. Although the mobilitiesof RDEC-1 EspA and EspB in SDS-PAGE were slightly faster than that ofenteropathogenic E. coli, the calculated molecular masses of RDEC-1 EspAand EspB were greater than that of enteropathogenic E. coli.

[0129] The amount of the other secreted proteins were decreased in theEspA⁻, EspB⁻, EspA⁻/EspB⁻ strains when compared to wild type RDEC-1strain. Furthermore, the decrease of detectable secretion of the 40kilodalton and 39 kilodalton proteins in the EspA⁻/EspB⁻ strain isgreater than that found in EspA⁻ and EspB⁻ strains. Secretion ofenteropathogenic E. coli proteins, except EspC, are mediated by a typeIII secretion system encoded by the sep cluster. It is possible thattruncation of EspA or EspB by inserting a stop codon may interfere withthis secretion pathway or feedback regulation of this system, therebyaffecting secretion of the other type III dependent secreted proteins.

[0130] Esp proteins are needed for triggering of the host signaltransduction pathway. Enteropathogenic E. coli EspA and EspB proteinsinduce host signal transduction pathways resulting in accumulation oftyrosine phosphorylated proteins, cytoskeletal actin, and othercomponents beneath the adherent bacteria. To determine whether RDEC-1EspA and EspB trigger these events in HeLa cells, cytoskeletal actin addtyrosine-phosphorylated proteins were stained with rhodamine-phallodinand fluorescently labelled anti-phosphotyrosine antibody. Although thelevel of accumulation of cytoskeletal actin and tyrosine-phosphorylatedprotein beneath the attached RDEC-1 is lower than that ofenteropathogenic E. coli, these behaviors were indistinguishable to thatof enteropathogenic E. coli. By contrast, RDEC-1 EspA⁻, EspB⁻, andEspA⁻/EspB⁻ strains did not accumulate cytoskeletal act in or tyrosine-phosphorylated proteins beneath the attached bacteria. However, theback mutant strains AAF003 and AAF004 accumulated these proteins similarto the parental strains.

[0131] When plasmids containing enteropathogenic E. coli espA or espB orboth were introduced into the RDEC-1 EspA⁻, EspB⁻, and EspA⁻/EspB⁻strains, the accumulation of cytoskeletal actin andtyrosinephosphorylated proteins was also restored. However, when theenteropathogenic E. coli EspA⁻ strain, which still secretes EspB, wascoinfected with RDEC-1 EspB, induction of host signal transductionevents were not restored. Enteropathogenic E. coli EspB also did notcomplement RDEC-1 EspA in co-infection experiment. Therefore,functionally EspA and EspB are similar in RDEC-1 and enteropathogenic E.coli with respect to activating host signal transduction pathways,although both proteins need to be secreted by the same strain.

[0132] Tyrosine-phosphorylated Hp90 could be detected by immunoblottingwhen HeLa cells were infected with enteropathogenic E. coli.Tyrosine-phosphorylated Hp90 could not be detected with RDEC-1 infectedcells, even though tyrosine phosphorylated proteins could be observedunder adherent RDEC-1 bacterial cells by immunofluorescence.

[0133] Enterohemorrhagic E. coli does not induce tyrosinephosphorylation in HEp-2 and T84 cells as judged by immunofluorescencemicroscopy. The sequencing results showed that RDEC-1 EspB is moresimilar to that of enterohemorrhagic E. coli than enteropathogenic E.coli. These results in this Example show that the lower accumulation oftyrosine-phosphorylated proteins during RDEC-1 infection is due to loweradherence efficiency of RDEC-1 because of differences in adhesion levelsor heterogeneity of Esp proteins or both.

[0134] Adherence and invasion ability. Enteropathogenic E. coli EspA andEspB are not only involved in triggering of host signal transductionpathways, but also necessary for invasion in vitro. In order toinvestigate the role of RDEC-1 EspA and EspB in adherence and invasion,RDEC-1 esp mutant strains were compared to that of RDEC-1. The adherenceability of EspA⁻, EspB⁻, and EspA⁻/EspB⁻ strains were similar to that ofthe wild type RDEC-1 strain. indicating that adherence is independent ofEspA and EspB expression. Although the invasive ability of wild typeRDEC-1 was about ninety times lower than that of enteropathogenic E.coli, this ability was further decreased in the mutant strains EspA⁻,EspB⁻, and EspA⁻/EspB⁻. However, invasion was restored by back mutationstrains AAF002 and AAF003. These results show that RDEC-1 invasionability depends upon EspA and EspB.

[0135] To determine the ability of enteropathogenic E. coli EspA andEspB to complement the RDEC-1 mutants, various plasmids containing theenteropathogenic E. coli espA and espB genes were introduced into theRDEC-1 mutant strains, and invasion efficiencies compared to that ofwild type RDEC-1 strain. Interestingly, the invasion levels of AAF001ΔAB(RDEC-1 EspA⁻/EspB⁻) harboring pMWespAB (EPEC EspA+/EspB+) was fourtimes greater than that of wild type RDEC-1, even though the amount ofsecreted enteropathogenic E. coli EspA and EspB in AAF001ΔAB strain waslower than that normally found in RDEC-1. Therefore, the differentinvasion levels observed between RDEC-1 and enteropathogenic E. colistrains in HeLa cells can be attributed to the Esp proteins, andenteropathogenic E. coli EspA and EspB are more efficient at mediatinginvasion in this tissue culture model. Homology comparisons showed thatEspA was highly conserved in RDEC-1 and enteropathogenic E. coli, butEspB was more heterogeneous, showing that the difference of invasiveabilities between RDEC-1 and enteropathogenic E. coli may be due to theEspB protein. Interestingly, enterohemorrhagic E. coli 0157 adheres to,but does not invade into human ileocecal (HCT-8) epithelial cells.RDEC-1 EspB was more similar to that of enterohemorrhagic E. coli ratherthan enteropathogenic E. coli, perhaps emphasizing the role of EspB ininvasion. These findings strongly support that Esp heterogeneity affectsthe invasive ability of enteropathogenic E. coli, RDEC-1, andenterohemorrhagic E. coli.

[0136] EspD mutant affects EspA and EspB secretion. Enteropathogenic E.coli contains an open reading frame, espD, located between espA andespB. To confirm the role of the espD product in secretion, the plasmidpMWespD encoding enteropathogenic E. coli espA. ΔespD (frame shiftmutation at the BglII site), and espD was constructed and introducedinto the RDEC-1 double mutant strain, AAF001AAB. The amount ofenteropathogenic E. coli EspA and EspB secreted proteins in AAF001AAB[pMWespD] was lower than that in AAF001AAB [pMWespAB], which containsfragment encoding intact enteropathogenic E. coli espA, espD, and espBgenes. Furthermore invasion ability was also decreased. These resultsshow that disruption of espD affects secretion of enteropathogenic E.coli EspA and EspB proteins. In this Example, we showed that mutationsin espA and/or espB also reduced the amount of the other secretedproteins, probably due to their truncated products. Secretion levelswere more decreased in espA and espB double mutants when compared withespA or espB mutants. Thus, truncated enteropathogenic E. coli EspD mayaffect the secretion of EspA and EspB in AAF001AAB [pMW6espD] due tointerference in type III secretion system. Whether or not EspD isdirectly involved in this secretion system is still unclear.

[0137] Enteropathogenic E. coli and RDEC-1 secreted proteins are tightlycontrolled by temperatures, which correspond to their relevant host bodytemperatures. Temperature regulates the expression of enteropathogenicE. coli and enterohemorrhagic E. coli 413/89-1 secreted proteins. EspBexpression was greatly increased when the temperature was shifted from20° C. to 37° C. Because EspA and EspB proteins are regulated byappropriate host body temperatures, wild type enteropathogenic E. coliand RDEC-1 strains were inoculated into DMEM and the secreted proteinswere prepared following incubation at various temperatures, thenanalyzed by SDS-PAGE. Expression of enteropathogenic E. coli secretedproteins were visible at 33° C. and reached maximal secretion level at36° C. Expression was decreased at 39° C., and no secreted proteins wereseen at 42° C. In contrast, maximal expression of RDEC-1 secretedproteins occurred at 39° C. and these proteins were still expressed at42° C. These results show that the maximal expression of Esp proteins inenteropathogenic E. coli and RDEC-1 are triggered by their relevanthost's body temperature, human (37° C.) and rabbit (39° C.).

[0138] In conclusion, both proteins were needed to trigger host signaltransduction pathways and invasion. Complementation experiments usingenteropathogenic E. coli esp genes revealed that host signaltransduction events triggered by RDEC-1 and enteropathogenic E. coliappear to be mediated by the same secreted proteins. Finally, optimalexpression of RDEC-1 and enteropathogenic E. coli secreted proteinscorrelated with their natural host's body temperature. This explainstheir strict host specificity and the lack of enteropathogenic E. coliinfection in rabbits or other animals. Animal infection studies usingRDEC-1 espA and espB strains will provide information about the role ofthese secreted proteins in virulence, and may possibly be useful forvaccine studies.

EXAMPLE 8 Two Rabbit Enteropathogenic E. coli (RDEC-1) SecretedProteins, EspA and EspB, are Virulence Factors

[0139] The purpose of this Example is to demonstrate the role of EspAand EspB proteins in pathogenesis. To investigate the role of theseproteins in virulence, mutations in espA and espB were constructed inthe rabbit enteropathogenic E. coli strain, RDEC-1.

[0140] RDEC-1 and its espA and espB mutant strains were inoculated bythe orogastric route into young rabbits. Most RDEC-1 was found in thececum and colon one week postinfection. However, the number of eithermutant strain was greatly decreased in these tissues compared to theparent strain. RDEC-1 adhered specifically to the sacculus rotundas(follicle associated epithelium) and bacterial colonization was alsoobserved in the cecum, indicating that the sacculus rotundas in thececum is an important colonization site for this pathogen. The adherencelevels of the EspA⁻ and EspB⁻ strains to the sacculus rotundas were 70and 8000 times less than that of parent strain. These results show thatthe adherence ability and tissue tropism of RDEC-1 are dependent on thetwo Esp secreted proteins. Furthermore, EspB appears to play a morecritical role than EspA in bacterial colonization and pathogenesis. Thisis the first demonstration that the enteropathogenic E. coli secretedproteins, EspA and EspB, which are involved in triggering of host cellsignal transduction pathways, are also needed for colonization andvirulence.

[0141] Animal infections were performed as follows: Overnight bacterialcultures were collected by centrifugation and resuspended in one ml ofphosphate-buffered saline. New Zealand white rabbits (weight 1.0 to 1.6kg) were fasted overnight, then five ml of 2.5% sterile sodiumbicarbonate and one ml of RDEC-1 or espA or espB strains (2.5×10¹⁰) wereinoculated into the stomach using orogastric tubes. The same dosage ofbacteria was inoculated into each rabbit the following day.

[0142] Clinical assessments were performed as follows: Each rabbit wasweighed daily and fecal shedding of bacteria were collected by rectalswabs and from stool pellets. Rectal swabs were rolled over one half ofthe surface of MacConkey plates containing nalidixic acid. Five stoolpellets or same amount of liquid stool were collected from each rabbitand resuspended in three ml phosphate-buffered saline and 0.1 ml of eachstool suspension was plated onto MacConkey plate containing nalidixicacid. The growth of nalidixic resistant colonies was scored as follows:0, no growth; 1, widely spaced colonies; 2, closely spaced colonies; 3,confluent growth of colonies.

[0143] Sampling and preparation of tissue were performed as follows:Tissues were excised immediately following sacrifice by intravenousinjection of ketamine and overdosing with sodium phenobarbital.

[0144] The amount of bacterial colonization in intestinal tissues wasassayed as follows: The intestinal segments (10 cm), except cecum, weredoubly ligated at their proximal and distal ends, and dissected betweenthe double ligated parts, then flushed with 10 ml of ice-coldphosphate-buffered saline. One gram of viscous contents from the cecumwas added to 9 ml phosphate-buffered saline. The resultingphosphate-buffered saline suspensions were diluted and plated onMacConkey plates containing nalidixic acid.

[0145] The amount of bacterial adherence to intestinal tissues wasassayed as follows: Tissue samples were excised using a nine mm diametercork punch, washed three times with phosphate-buffered saline, added totwo ml of ice-cold phosphate-buffered saline, and homogenized with ahomogenizer, then serial diluted samples were plated onto MacConkeyplates. The numbers of bacteria adherent to each tissue per squarecentimeter were calculated as follows: CFU/cm2=the bacterialnumber/plate×dilution factor×2 ml/˜0.452.

EXAMPLE 9 Development of an Assay to Screen for Inhibitors of BacterialType III Secretion

[0146] The purpose of this Example is to provide an assay to screen forinhibitors of bacterial type III secretion.

[0147] A polynucleotide encoding the EspA polypeptide is fused toseveral well known molecules, including a HSV tag. The gene fusion isstill secreted out of enteropathogenic E. coli. A plasmid contains thegenetic region of espA that encodes the amino-terminal portion of EspA(needed to mediate type III secretion) fused to a Herpes Simplex Virus(HSV) sequence that encodes a sequence tag to which commercialantibodies are available. This plasmid is transformed into a strain thatcontains an espA mutation yet still secretes the other enteropathogenicE. coli-secreted proteins that use the type III secretion system. Thesupernatant of organisms containing these fusions is collected, added toan ELISA plate, followed by standard ELISA. A calorimetric readoutindicates the fusion protein is secreted.

[0148] This plasmid is also transformed into a strain which is defectivefor type III secretion (i e. a negative control). When the fusionprotein is expressed in this strain, the fusion protein is expressed butnot secreted. ELISA results with this mutant confirm that it is notsecreted.

[0149] Thus an easy ELISA assay to look at secretion is provided. Thisassay is simple, and requires no special technology. It is alsoeconomical, because no expensive reagents are needed. It is automatedand used to screen reagents to identify inhibitors of bacterial type IIIsecretion.

[0150] To assay for secretion, bacteria are grown standing overnight intissue culture fluid in the presence of compounds to be tested. Theseconditions yield enteropathogenic E. coli-mediated secretion. Thefollowing day, bacteria are removed by centrifugation, and thesupernatant placed into wells of a 96 well microtiter plate. A standardELISA is performed on the supernatants. If the compound being tested isbactericidal, the bacteria do not grow overnight.

[0151] A polynucleotide encoding another polypeptide secreted byenteropathogenic E. coli is fused to several well known molecules. Apolynucleotide encoding EspB polypeptide was fused a HSV tag, to whichcommercial antibodies are available. The gene fusion was still secretedout of enteropathogenic E. coli. This plasmid was transformed into astrain that contained an espA mutation yet still secretes the otherenteropathogenic E. coli-secreted proteins that use the type IIIsecretion system. The supernatant of organisms containing these fusionsis collected, added to an ELISA plate, followed by standard ELISAtechnology with, for example, anti-HSV antibodies. This screen was usedto assay plant extracts from medically important plants. Dilutions of1/200-1/1000 (about 250 μg/ml) are appropriate. Promising compounds arerescreened in the ELISA secretion assay to check for reproducibility.

[0152] Although the invention has been described with reference to thepresently preferred embodiments, it should be understood that variousmodifications can be made without departing from the spirit of theinvention. Accordingly, the invention is limited only by the followingclaims.

1 15 1 639 DNA Escherichia coli CDS (46)..(624) 1 ttatagtttt tgtcatgctaagaaagatta tgaagaggta tatac atg gat aca tca 57 Met Asp Thr Ser 1 act acagca tca gtt gct agt gcg aat gcg agt act tcg aca tca atg 105 Thr Thr AlaSer Val Ala Ser Ala Asn Ala Ser Thr Ser Thr Ser Met 5 10 15 20 gcc tatgat tta ggg agc atg tcg aaa gat gac gtt att gat cta ttt 153 Ala Tyr AspLeu Gly Ser Met Ser Lys Asp Asp Val Ile Asp Leu Phe 25 30 35 aat aaa ctcggt gtt ttt cag gct gca att ctc atg ttt gcc tat atg 201 Asn Lys Leu GlyVal Phe Gln Ala Ala Ile Leu Met Phe Ala Tyr Met 40 45 50 tat cag gca caaagc gat ctg tcg att gca aag ttt gct gat atg aat 249 Tyr Gln Ala Gln SerAsp Leu Ser Ile Ala Lys Phe Ala Asp Met Asn 55 60 65 gag gca tct aag gagtca acc act gcc caa aaa atg gct aat ctt gta 297 Glu Ala Ser Lys Glu SerThr Thr Ala Gln Lys Met Ala Asn Leu Val 70 75 80 gat gct aaa att gct gacgtt cag agt agc tct gac aag aat gcg aaa 345 Asp Ala Lys Ile Ala Asp ValGln Ser Ser Ser Asp Lys Asn Ala Lys 85 90 95 100 gct caa ctt cct gat gaagtg att tca tat ata aat gat cct cgc aat 393 Ala Gln Leu Pro Asp Glu ValIle Ser Tyr Ile Asn Asp Pro Arg Asn 105 110 115 gac att aca ata agt ggtatt gac aat ata aat gct caa tta ggc gct 441 Asp Ile Thr Ile Ser Gly IleAsp Asn Ile Asn Ala Gln Leu Gly Ala 120 125 130 ggt gat ttg caa acg gtgaaa gca gct att tca gct aaa gcg aat aat 489 Gly Asp Leu Gln Thr Val LysAla Ala Ile Ser Ala Lys Ala Asn Asn 135 140 145 ttg aca acg acg gtc aataat agc cag ctt gaa ata cag caa atg tca 537 Leu Thr Thr Thr Val Asn AsnSer Gln Leu Glu Ile Gln Gln Met Ser 150 155 160 aat acg tta aac cta ttaacg agt gca cgt tct gat atg cag tca ctg 585 Asn Thr Leu Asn Leu Leu ThrSer Ala Arg Ser Asp Met Gln Ser Leu 165 170 175 180 caa tat aga act atttca gga ata tcc ctt ggt aaa taa ccggacataa 634 Gln Tyr Arg Thr Ile SerGly Ile Ser Leu Gly Lys 185 190 ctatg 639 2 192 PRT Escherichia coli 2Met Asp Thr Ser Thr Thr Ala Ser Val Ala Ser Ala Asn Ala Ser Thr 1 5 1015 Ser Thr Ser Met Ala Tyr Asp Leu Gly Ser Met Ser Lys Asp Asp Val 20 2530 Ile Asp Leu Phe Asn Lys Leu Gly Val Phe Gln Ala Ala Ile Leu Met 35 4045 Phe Ala Tyr Met Tyr Gln Ala Gln Ser Asp Leu Ser Ile Ala Lys Phe 50 5560 Ala Asp Met Asn Glu Ala Ser Lys Glu Ser Thr Thr Ala Gln Lys Met 65 7075 80 Ala Asn Leu Val Asp Ala Lys Ile Ala Asp Val Gln Ser Ser Ser Asp 8590 95 Lys Asn Ala Lys Ala Gln Leu Pro Asp Glu Val Ile Ser Tyr Ile Asn100 105 110 Asp Pro Arg Asn Asp Ile Thr Ile Ser Gly Ile Asp Asn Ile AsnAla 115 120 125 Gln Leu Gly Ala Gly Asp Leu Gln Thr Val Lys Ala Ala IleSer Ala 130 135 140 Lys Ala Asn Asn Leu Thr Thr Thr Val Asn Asn Ser GlnLeu Glu Ile 145 150 155 160 Gln Gln Met Ser Asn Thr Leu Asn Leu Leu ThrSer Ala Arg Ser Asp 165 170 175 Met Gln Ser Leu Gln Tyr Arg Thr Ile SerGly Ile Ser Leu Gly Lys 180 185 190 3 726 DNA Escherichia coli CDS(103)..(681) 3 ttaatgattg gtaaagtaat tgattataag gaggatgtta tttgatattggttttttaat 60 cgtttttggt cttgctaaga aagattatta agaggtatat ac atg gat acatca 114 Met Asp Thr Ser 1 act gca aca tca gtt gct agt gcg aac gcg agtact tcg aca tcg aca 162 Thr Ala Thr Ser Val Ala Ser Ala Asn Ala Ser ThrSer Thr Ser Thr 5 10 15 20 gtc tat gac tta ggc agt atg tcg aaa gac gaagta gtt cag cta ttt 210 Val Tyr Asp Leu Gly Ser Met Ser Lys Asp Glu ValVal Gln Leu Phe 25 30 35 aat aaa gtc ggt gtt ttt cag gct gcg ctt ctc atgttt gcc tat atg 258 Asn Lys Val Gly Val Phe Gln Ala Ala Leu Leu Met PheAla Tyr Met 40 45 50 tat cag gca caa agc gat ctg tcg att gca aag ttt gctgat atg aat 306 Tyr Gln Ala Gln Ser Asp Leu Ser Ile Ala Lys Phe Ala AspMet Asn 55 60 65 gag gca tct aag gag tca acc aca gcc caa aaa atg gct aatctt gtg 354 Glu Ala Ser Lys Glu Ser Thr Thr Ala Gln Lys Met Ala Asn LeuVal 70 75 80 gat gct aaa att gct gat gtt cag agt agt tct gac aag aat aagaaa 402 Asp Ala Lys Ile Ala Asp Val Gln Ser Ser Ser Asp Lys Asn Lys Lys85 90 95 100 gcc aaa ctt cct caa gaa gtg att gac tat ata aat gat cct cgcaat 450 Ala Lys Leu Pro Gln Glu Val Ile Asp Tyr Ile Asn Asp Pro Arg Asn105 110 115 gac att aca gta agt ggt att agc gat cta aat gct gaa tta ggcgct 498 Asp Ile Thr Val Ser Gly Ile Ser Asp Leu Asn Ala Glu Leu Gly Ala120 125 130 ggt gat ttg caa acg gtg aag gcc gct att tcg gcc aaa tcg aataac 546 Gly Asp Leu Gln Thr Val Lys Ala Ala Ile Ser Ala Lys Ser Asn Asn135 140 145 ttg acc acg gta gtg aat aat agc cag ctt gaa ata cag caa atgtca 594 Leu Thr Thr Val Val Asn Asn Ser Gln Leu Glu Ile Gln Gln Met Ser150 155 160 aat acg tta aac cta tta acg agt gca cgt tct gat att cag tcactg 642 Asn Thr Leu Asn Leu Leu Thr Ser Ala Arg Ser Asp Ile Gln Ser Leu165 170 175 180 caa tac aga act att tca gca ata tcc ctt ggt aaa taaccggagataa 691 Gln Tyr Arg Thr Ile Ser Ala Ile Ser Leu Gly Lys 185 190ctatgcttaa tgtaaatagc gatatccagt ctatg 726 4 192 PRT Escherichia coli 4Met Asp Thr Ser Thr Ala Thr Ser Val Ala Ser Ala Asn Ala Ser Thr 1 5 1015 Ser Thr Ser Thr Val Tyr Asp Leu Gly Ser Met Ser Lys Asp Glu Val 20 2530 Val Gln Leu Phe Asn Lys Val Gly Val Phe Gln Ala Ala Leu Leu Met 35 4045 Phe Ala Tyr Met Tyr Gln Ala Gln Ser Asp Leu Ser Ile Ala Lys Phe 50 5560 Ala Asp Met Asn Glu Ala Ser Lys Glu Ser Thr Thr Ala Gln Lys Met 65 7075 80 Ala Asn Leu Val Asp Ala Lys Ile Ala Asp Val Gln Ser Ser Ser Asp 8590 95 Lys Asn Lys Lys Ala Lys Leu Pro Gln Glu Val Ile Asp Tyr Ile Asn100 105 110 Asp Pro Arg Asn Asp Ile Thr Val Ser Gly Ile Ser Asp Leu AsnAla 115 120 125 Glu Leu Gly Ala Gly Asp Leu Gln Thr Val Lys Ala Ala IleSer Ala 130 135 140 Lys Ser Asn Asn Leu Thr Thr Val Val Asn Asn Ser GlnLeu Glu Ile 145 150 155 160 Gln Gln Met Ser Asn Thr Leu Asn Leu Leu ThrSer Ala Arg Ser Asp 165 170 175 Ile Gln Ser Leu Gln Tyr Arg Thr Ile SerAla Ile Ser Leu Gly Lys 180 185 190 5 989 DNA Escherichia coli CDS(31)..(975) 5 tcgagtttaa ttattaaaga gaatttaatt atg aat act att gat tatact aat 54 Met Asn Thr Ile Asp Tyr Thr Asn 1 5 caa gta atg acg gtt aattct gtt tcg gag aat act acc ggc tct aat 102 Gln Val Met Thr Val Asn SerVal Ser Glu Asn Thr Thr Gly Ser Asn 10 15 20 gca att acc gca tct gct attaat tca tct ttg ctt acc gat ggt aag 150 Ala Ile Thr Ala Ser Ala Ile AsnSer Ser Leu Leu Thr Asp Gly Lys 25 30 35 40 gtc gat gtt tct aaa ctg atgctg gaa att caa aaa ctc ctg ggc aag 198 Val Asp Val Ser Lys Leu Met LeuGlu Ile Gln Lys Leu Leu Gly Lys 45 50 55 atg gtg cgt ata ttg cag gat taccaa cag caa cag ttg tcg cag agc 246 Met Val Arg Ile Leu Gln Asp Tyr GlnGln Gln Gln Leu Ser Gln Ser 60 65 70 tat cag atc caa ctg gcc gtt ttt gagagc cag aat aaa gcc att gat 294 Tyr Gln Ile Gln Leu Ala Val Phe Glu SerGln Asn Lys Ala Ile Asp 75 80 85 gaa aaa aag gcc gct gca aca gcc gct ctggtt ggt ggg gct att tca 342 Glu Lys Lys Ala Ala Ala Thr Ala Ala Leu ValGly Gly Ala Ile Ser 90 95 100 tca gta ttg ggg atc tta ggc tct ttt gcagca att aac agt gct acg 390 Ser Val Leu Gly Ile Leu Gly Ser Phe Ala AlaIle Asn Ser Ala Thr 105 110 115 120 aaa ggc gcg agt gat att gct caa aaaacc gcc tct aca tct tct aag 438 Lys Gly Ala Ser Asp Ile Ala Gln Lys ThrAla Ser Thr Ser Ser Lys 125 130 135 gct att gat gcg gct tct gat act gcgact aaa acg ttg act aag gca 486 Ala Ile Asp Ala Ala Ser Asp Thr Ala ThrLys Thr Leu Thr Lys Ala 140 145 150 acg gaa agc gtt gct gat gct gtt gaagat gca tcc agc gtg atg cag 534 Thr Glu Ser Val Ala Asp Ala Val Glu AspAla Ser Ser Val Met Gln 155 160 165 caa gcg atg act aca gca acg aga gcggcc agc cgt aca tcc gac gtt 582 Gln Ala Met Thr Thr Ala Thr Arg Ala AlaSer Arg Thr Ser Asp Val 170 175 180 gct gat gac att gcc gat tct gct cagaga gct tct cag ctg gct gaa 630 Ala Asp Asp Ile Ala Asp Ser Ala Gln ArgAla Ser Gln Leu Ala Glu 185 190 195 200 aac gct gca gat gcc gct cag aaggca agt cgg gca agc cgc ttt atg 678 Asn Ala Ala Asp Ala Ala Gln Lys AlaSer Arg Ala Ser Arg Phe Met 205 210 215 gct gca gta gat aag att act ggctct aca cca ttt att gcc gtt acc 726 Ala Ala Val Asp Lys Ile Thr Gly SerThr Pro Phe Ile Ala Val Thr 220 225 230 agt ctt gcc gaa ggc acg aag acattg cca aca acg gta tct gaa tca 774 Ser Leu Ala Glu Gly Thr Lys Thr LeuPro Thr Thr Val Ser Glu Ser 235 240 245 gtc aaa tct aac cat gag att agcgaa cag cgt tat aag tct gtg gag 822 Val Lys Ser Asn His Glu Ile Ser GluGln Arg Tyr Lys Ser Val Glu 250 255 260 aac ttc cag cag ggt aat ttg gatctg tat aag caa gaa gtt cgc aga 870 Asn Phe Gln Gln Gly Asn Leu Asp LeuTyr Lys Gln Glu Val Arg Arg 265 270 275 280 gcg cag gat gat atc gct agccgt ctg cgt gat atg aca aca gcc gct 918 Ala Gln Asp Asp Ile Ala Ser ArgLeu Arg Asp Met Thr Thr Ala Ala 285 290 295 cgc gat ctc act gat ctt cagaat cgt atg ggt caa tcg gtt cgc tta 966 Arg Asp Leu Thr Asp Leu Gln AsnArg Met Gly Gln Ser Val Arg Leu 300 305 310 gct ggg taa ttgatcatgg tcga989 Ala Gly 6 314 PRT Escherichia coli 6 Met Asn Thr Ile Asp Tyr Thr AsnGln Val Met Thr Val Asn Ser Val 1 5 10 15 Ser Glu Asn Thr Thr Gly SerAsn Ala Ile Thr Ala Ser Ala Ile Asn 20 25 30 Ser Ser Leu Leu Thr Asp GlyLys Val Asp Val Ser Lys Leu Met Leu 35 40 45 Glu Ile Gln Lys Leu Leu GlyLys Met Val Arg Ile Leu Gln Asp Tyr 50 55 60 Gln His Gln Gln Leu Ser GlnSer Tyr Gln Ile Gln Leu Ala Val Phe 65 70 75 80 Glu Ser Gln Asn Lys AlaIle Asp Glu Lys Lys Ala Ala Ala Thr Ala 85 90 95 Ala Leu Val Gly Gly AlaIle Ser Ser Val Leu Gly Ile Leu Gly Ser 100 105 110 Phe Ala Ala Ile AsnSer Ala Thr Lys Gly Ala Ser Asp Ile Ala Gln 115 120 125 Lys Thr Ala SerThr Ser Ser Lys Ala Ile Asp Ala Ala Ser Asp Thr 130 135 140 Ala Thr LysThr Leu Thr Lys Ala Thr Glu Ser Val Ala Asp Ala Val 145 150 155 160 GluAsp Ala Ser Ser Val Met Gln Gln Ala Met Thr Thr Ala Thr Arg 165 170 175Ala Ala Ser Arg Thr Ser Asp Val Ala Asp Asp Ile Ala Asp Ser Ala 180 185190 Gln Arg Ala Ser Gln Leu Ala Glu Asn Ala Ala Asp Ala Ala Gln Lys 195200 205 Ala Ser Arg Ala Ser Arg Phe Met Ala Ala Val Asp Lys Ile Thr Gly210 215 220 Ser Thr Pro Phe Ile Ala Val Thr Ser Leu Ala Glu Gly Thr LysThr 225 230 235 240 Leu Pro Thr Thr Val Ser Glu Ser Val Lys Ser Asn HisGlu Ile Ser 245 250 255 Glu Gln Arg Tyr Lys Ser Val Glu Asn Phe Gln GlnGly Asn Leu Asp 260 265 270 Leu Tyr Lys Gln Glu Val Arg Arg Ala Gln AspAsp Ile Ala Ser Arg 275 280 285 Leu Arg Asp Met Thr Thr Ala Ala Pro AspLeu Thr Asp Leu Gln Asn 290 295 300 Arg Met Gly Gln Ser Val Arg Leu AlaGly 305 310 7 194 PRT Escherichia coli 7 Met Asp Thr Ser Thr Ala Thr SerVal Ala Ser Ala Asn Ala Ser Thr 1 5 10 15 Ser Thr Ser Thr Val Tyr AspLeu Gly Ser Met Ser Lys Asp Glu Val 20 25 30 Val Gln Leu Phe Asn Lys ValGly Val Phe Gln Ala Ala Leu Leu Met 35 40 45 Phe Ala Tyr Met Tyr Gln AlaGln Ser Asp Leu Ser Ile Ala Lys Phe 50 55 60 Ala Asp Met Asn Glu Ala SerLys Glu Ser Thr Thr Ala Gln Lys Met 65 70 75 80 Ala Asn Leu Val Asp AlaLys Ile Ala Asp Val Gln Ser Ser Ser Asp 85 90 95 Lys Asn Lys Lys Ala LysLeu Pro Gln Glu Val Ile Asp Tyr Ile Asn 100 105 110 Asp Pro Arg Asn AspIle Thr Val Ser Gly Ile Ser Asp Leu Asn Ala 115 120 125 Glu Leu Gly AlaGly Ala Gly Asp Leu Gln Thr Val Lys Ala Ala Ile 130 135 140 Ser Ala LysSer Asn Asn Leu Thr Thr Val Val Asn Asn Ser Gln Leu 145 150 155 160 GluIle Gln Gln Met Ser Asn Thr Leu Asn Leu Leu Thr Ser Ala Arg 165 170 175Ser Asp Ile Gln Ser Leu Gln Tyr Arg Thr Ile Ser Ala Ile Ser Leu 180 185190 Gly Lys 8 194 PRT Escherichia coli 8 Met Asp Thr Ser Thr Thr Ala SerVal Ala Ser Ala Asn Ala Ser Thr 1 5 10 15 Ser Thr Ser Met Ala Tyr AspLeu Gly Ser Met Ser Lys Asp Asp Val 20 25 30 Ile Asp Leu Phe Asn Lys LeuGly Val Phe Gln Ala Ala Ile Leu Met 35 40 45 Phe Ala Tyr Met Tyr Gln AlaGln Ser Asp Leu Ser Ile Ala Lys Phe 50 55 60 Ala Asp Met Asn Glu Ala SerLys Glu Ser Thr Thr Ala Gln Lys Met 65 70 75 80 Ala Asn Leu Val Asp AlaLys Ile Ala Asp Val Gln Ser Ser Ser Asp 85 90 95 Lys Asn Ala Lys Ala GlnLeu Pro Asp Glu Val Ile Ser Tyr Ile Asn 100 105 110 Asp Pro Arg Asn AspIle Thr Ile Ser Gly Ile Asp Asn Ile Asn Ala 115 120 125 Gln Leu Gly AlaGly Ala Gly Asp Leu Gln Thr Val Lys Ala Ala Ile 130 135 140 Ser Ala LysAla Asn Asn Leu Thr Thr Thr Val Asn Asn Ser Gln Leu 145 150 155 160 GluIle Gln Gln Met Ser Asn Thr Leu Asn Leu Leu Thr Ser Ala Arg 165 170 175Ser Asp Met Gln Ser Leu Gln Tyr Arg Thr Ile Ser Gly Ile Ser Leu 180 185190 Gly Lys 9 314 PRT Escherichia coli 9 Met Asn Thr Ile Asp Tyr Thr AsnGln Val Met Thr Val Asn Ser Val 1 5 10 15 Ser Glu Asn Thr Thr Gly SerAsn Ala Ile Thr Ala Ser Ala Ile Asn 20 25 30 Ser Ser Leu Leu Thr Asp GlyLys Val Asp Val Ser Lys Leu Met Leu 35 40 45 Glu Ile Gln Lys Leu Leu GlyLys Met Val Arg Ile Leu Gln Asp Tyr 50 55 60 Gln Gln Gln Gln Leu Ser GlnSer Tyr Gln Ile Gln Leu Ala Val Phe 65 70 75 80 Glu Ser Gln Asn Lys AlaIle Asp Glu Lys Lys Ala Ala Ala Thr Ala 85 90 95 Ala Leu Val Gly Gly AlaIle Ser Ser Val Leu Gly Ile Leu Gly Ser 100 105 110 Phe Ala Ala Ile AsnSer Ala Thr Lys Gly Ala Ser Asp Ile Ala Gln 115 120 125 Lys Thr Ala SerThr Ser Ser Lys Ala Ile Asp Ala Ala Ser Asp Thr 130 135 140 Ala Thr LysThr Leu Thr Lys Ala Thr Glu Ser Val Ala Asp Ala Val 145 150 155 160 GluAsp Ala Ser Ser Val Met Gln Gln Ala Met Thr Thr Ala Thr Arg 165 170 175Ala Ala Ser Arg Thr Ser Asp Val Ala Asp Asp Ile Ala Asp Ser Ala 180 185190 Gln Arg Ala Ser Gln Leu Ala Glu Asn Ala Ala Asp Ala Ala Gln Lys 195200 205 Ala Ser Arg Ala Ser Arg Phe Met Ala Ala Val Asp Lys Ile Thr Gly210 215 220 Ser Thr Pro Phe Ile Ala Val Thr Ser Leu Ala Glu Gly Thr LysThr 225 230 235 240 Leu Pro Thr Thr Val Ser Glu Ser Val Lys Ser Asn HisGlu Ile Ser 245 250 255 Glu Gln Arg Tyr Lys Ser Val Glu Asn Phe Gln GlnGly Asn Leu Asp 260 265 270 Leu Tyr Lys Gln Glu Val Arg Arg Ala Gln AspAsp Ile Ala Ser Arg 275 280 285 Leu Arg Asp Met Thr Thr Ala Ala Arg AspLeu Thr Asp Leu Gln Asn 290 295 300 Arg Met Gly Gln Ser Val Arg Leu AlaGly 305 310 10 314 PRT Escherichia coli 10 Met Asn Thr Ile Asp Tyr ThrAsn Gln Val Met Thr Val Asn Ser Val 1 5 10 15 Ser Glu Asn Thr Thr GlySer Asn Ala Ile Thr Ala Ser Ala Ile Asn 20 25 30 Ser Ser Leu Leu Thr AspGly Lys Val Asp Val Ser Lys Leu Met Leu 35 40 45 Glu Ile Gln Lys Leu LeuGly Lys Met Val Arg Ile Leu Gln Asp Tyr 50 55 60 Gln Gln Gln Gln Leu SerGln Ser Tyr Gln Ile Gln Leu Ala Val Phe 65 70 75 80 Glu Ser Gln Asn LysAla Ile Asp Glu Lys Lys Ala Ala Ala Thr Ala 85 90 95 Ala Leu Val Gly GlyAla Ile Ser Ser Val Leu Gly Ile Leu Gly Ser 100 105 110 Phe Ala Ala IleAsn Ser Ala Thr Lys Gly Ala Ser Asp Ile Ala Gln 115 120 125 Lys Thr AlaSer Thr Ser Ser Lys Ala Ile Asp Ala Ala Ser Asp Thr 130 135 140 Ala ThrLys Thr Leu Thr Lys Ala Thr Glu Ser Val Ala Asp Ala Val 145 150 155 160Glu Asp Ala Ser Ser Val Met Gln Gln Ala Met Thr Thr Ala Thr Arg 165 170175 Ala Ala Ser Arg Thr Ser Asp Val Ala Asp Asp Ile Ala Asp Ser Ala 180185 190 Gln Arg Ala Ser Gln Leu Ala Glu Asn Ala Ala Asp Ala Ala Gln Lys195 200 205 Ala Ser Arg Ala Ser Arg Phe Met Ala Ala Val Asp Lys Ile ThrGly 210 215 220 Ser Thr Pro Phe Ile Ala Val Thr Ser Leu Ala Glu Gly ThrLys Thr 225 230 235 240 Leu Pro Thr Thr Val Ser Glu Ser Val Lys Ser AsnHis Glu Ile Ser 245 250 255 Glu Gln Arg Tyr Lys Ser Val Glu Asn Phe GlnGln Gly Asn Leu Asp 260 265 270 Leu Tyr Lys Gln Glu Val Arg Arg Ala GlnAsp Asp Ile Ala Ser Arg 275 280 285 Leu Arg Asp Met Thr Thr Ala Ala ArgAsp Leu Thr Asp Leu Gln Asn 290 295 300 Arg Met Gly Gln Ser Val Arg LeuAla Gly 305 310 11 312 PRT Escherichia coli 11 Met Asn Thr Ile Asp AsnThr Gln Val Thr Met Val Asn Ser Ala Ser 1 5 10 15 Glu Ser Thr Thr GlyAla Ser Ser Ala Val Ala Ala Ser Ala Leu Ser 20 25 30 Ile Asp Ser Ser LeuLeu Thr Asp Gly Lys Val Asp Ile Cys Lys Leu 35 40 45 Met Leu Glu Ile GlnLys Leu Leu Gly Lys Met Val Thr Leu Leu Gln 50 55 60 Asp Tyr Gln Gln LysGln Leu Ala Gln Ser Tyr Gln Ile Gln Gln Ala 65 70 75 80 Val Phe Glu SerGln Asn Lys Ala Ile Glu Glu Lys Lys Ala Ala Ala 85 90 95 Thr Ala Ala LeuVal Gly Gly Ile Ile Ser Ser Ala Leu Gly Ile Leu 100 105 110 Gly Ser PheAla Ala Met Asn Asn Ala Ala Lys Gly Ala Gly Glu Ile 115 120 125 Ala GluLys Ala Ser Ser Ala Ser Ser Lys Ala Ala Gly Ala Ala Ser 130 135 140 GluVal Ala Asn Lys Ala Leu Val Lys Ala Thr Glu Ser Val Ala Asp 145 150 155160 Val Ala Glu Glu Ala Ser Ser Ala Met Gln Lys Ala Met Ala Thr Thr 165170 175 Thr Lys Ala Ala Ser Arg Ala Ser Gly Val Ala Asp Asp Val Ala Lys180 185 190 Ala Thr Asp Phe Ala Glu Asp Leu Ala Asp Ala Ala Glu Lys ThrSer 195 200 205 Arg Ile Asn Lys Leu Leu Asn Ser Val Asp Lys Leu Thr AsnThr Thr 210 215 220 Ala Phe Val Ala Val Thr Ser Leu Ala Glu Gly Thr LysThr Leu Pro 225 230 235 240 Thr Thr Ile Ser Glu Ser Val Lys Ser Thr HisGlu Val Asn Glu Gln 245 250 255 Arg Ala Lys Ser Leu Glu Asn Phe Gln GlnGly Asn Leu Glu Leu Tyr 260 265 270 Lys Gln Asp Val Arg Arg Thr Gln AspAsp Ile Thr Thr Arg Leu Arg 275 280 285 Asp Ile Thr Ser Ala Val Arg AspLeu Leu Glu Val Gln Asn Arg Met 290 295 300 Gly Gln Ser Gly Arg Leu AlaGly 305 310 12 321 PRT Escherichia coli 12 Met Asn Thr Ile Asp Asn AsnAsn Ala Ala Ile Ala Val Asn Ser Val 1 5 10 15 Leu Ser Ser Thr Thr AspSer Thr Ser Ser Thr Thr Thr Ser Ala Ser 20 25 30 Ser Ile Ser Ser Ser LeuLeu Thr Asp Gly Arg Val Asp Ile Ser Lys 35 40 45 Leu Met Leu Glu Val GlnLys Leu Leu Arg Glu Met Val Thr Thr Leu 50 55 60 Gln Asp Tyr Leu Gln LysGln Leu Ala Gln Ser Tyr Asp Ile Gln Lys 65 70 75 80 Ala Val Phe Glu SerGln Asn Lys Ala Ile Asp Glu Lys Lys Ala Gly 85 90 95 Ala Thr Ala Ala LeuIle Gly Gly Ala Ile Ser Ser Val Leu Gly Ile 100 105 110 Leu Gly Ser PheAla Ala Ile Asn Ser Ala Thr Lys Gly Ala Ser Asp 115 120 125 Val Ala GlnGln Ala Ala Ser Thr Ser Ala Lys Ser Ile Gly Thr Val 130 135 140 Ser GluAla Ser Thr Lys Ala Leu Ala Lys Ala Ser Glu Gly Ile Ala 145 150 155 160Asp Ala Ala Asp Asp Ala Ala Gly Ala Met Gln Gln Thr Ile Ala Thr 165 170175 Ala Ala Lys Ala Ala Ser Arg Thr Ser Gly Ile Thr Asp Asp Val Ala 180185 190 Thr Ser Ala Gln Lys Ala Ser Gln Val Ala Glu Glu Ala Ala Asp Ala195 200 205 Ala Gln Glu Leu Ala Gln Lys Ala Gly Leu Leu Ser Arg Phe MetAla 210 215 220 Ala Ala Gly Arg Ile Ser Gly Ser Thr Pro Phe Ile Val ValThr Ser 225 230 235 240 Leu Ala Glu Gly Thr Lys Thr Leu Pro Thr Thr IleSer Glu Ser Val 245 250 255 Lys Ser Asn His Asp Ile Asn Glu Gln Arg AlaLys Ser Val Glu Asn 260 265 270 Leu Gln Ala Ser Asn Leu Asp Leu Tyr LysGln Asp Val Arg Arg Ala 275 280 285 Gln Asp Asp Ile Ser Ser Arg Leu ArgAsp Met Thr Thr Thr Ala Arg 290 295 300 Asp Leu Thr Asp Leu Ile Asn ArgMet Gly Gln Ala Ala Arg Leu Ala 305 310 315 320 Gly 13 11 PRTEscherichia coli 13 Met Leu Asn Val Asn Ser Asp Ile Gln Ser Met 1 5 1014 50 PRT Escherichia coli 14 Met Leu Asn Val Asn Asn Asp Ile Gln SerVal Arg Ser Gly Ala Ser 1 5 10 15 Ala Ala Thr Ala Thr Ser Gly Ile AsnGln Ser Glu Val Thr Ser Ala 20 25 30 Leu Asp Leu Gln Leu Val Lys Ser ThrAla Pro Ser Ala Ser Trp Thr 35 40 45 Glu Ser 50 15 856 DNA Escherichiacoli 15 ccttcggggt gactaaacta aactaggagg aataaatggc taaaatgagaatatcaccgg 60 aattgaaaaa actgatcgaa aaataccgct gcgtaaaaga tacggaaggaatgtctcctg 120 ctaaggtata taagctggtg ggagaaaatg aaaacctata tttaaaaatgacggacagcc 180 ggtataaagg gaccacctat gatgtggaac gggaaaagga catgatgctatggctggaag 240 gaaagctgcc tgttccaaag gtcctgcact ttgaacggca tgatggctggagcaatctgc 300 tcatgagtga ggccgatggc gtcctttgct cggaagagta tgaagatgaacaaagccctg 360 aaaagattat cgagctgtat gcggagtgca tcaggctctt tcactccatcgacatatcgg 420 attgtcccta tacgaatagc ttagacagcc gcttagccga attggattacttactgaata 480 acgatctggc cgatgtggat tgcgaaaact gggaagaaga cactccatttaaagatccgc 540 gcgagctgta tgatttttta aagacggaaa agcccgaaga ggaacttgtcttttcccacg 600 gcgacctggg agacagcaac atctttgtga aagatggcaa agtaagtggctttattgatc 660 ttgggagaag cggcagggcg gacaagtggt atgacattgc cttctgcgtccggtcgatca 720 gggaggatat cggggaagaa cagtatgtcg agctattttt tgacttactggggatcaagc 780 ctgattggga gaaaataaaa tattatattt tactggatga attgttttagtacctggagg 840 aataatgacc ccgacg 856

We claim:
 1. An isolated EspA polypeptide characterized by: a) being asecreted protein from enteropathogenic or enterohemorrhagic E. coli; andb) comprising an amino acid sequence as set forth in SEQ ID NO:2 or SEQID NO:4.
 2. An isolated polynucleotide encoding the polypeptide ofclaim
 1. 3. An isolated polynucleotide selected from the groupconsisting of: a) the nucleic acid sequence set forth in SEQ ID NO: 1;b) the nucleic acid sequence set forth in SEQ ID NO: 1, wherein T is U;c) nucleic acid sequences complementary to a); and d) fragments of a),b) or c) that are at least 15 nucleotide bases in length and thathybridize under stringent conditions to DNA which encodes thepolypeptide set forth in SEQ ID NO:
 2. 4. An isolated polynucleotideselected from the group consisting of: a) the nucleic acid sequence setforth in SEQ ID NO: 3; b) the nucleic acid sequence set forth in SEQ IDNO: 3, wherein T is U; c) nucleic acid sequences complementary to a);and d) fragments of a), b) or c) that are at least 15 nucleotide basesin length and that hybridize under stringent conditions to DNA whichencodes the polypeptide set forth in SEQ ID NO:
 4. 5. A nucleic acidexpression vector comprising a promoter operably linked to thepolynucleotide of claim
 2. 6. A host cell containing the vector of claim5.
 7. An antibody specific for the polypeptide of claim
 1. 8. Theantibody of claim 7, wherein the antibody is monoclonal.
 9. The antibodyof claim 7, wherein the antibody is polyclonal.
 10. A method fordetecting EspA polypeptide in a sample, comprising: a) contacting thesample with the antibody of claim 7; and b) detecting binding of theantibody of claim 7 to EspA polypeptide, wherein binding is indicativeof the presence of EspA polypeptide in the sample.
 11. The method ofclaim 10, wherein the sample is tissue.
 12. The method of claim 10,wherein the sample is a biological fluid.
 13. The method of claim 10,wherein the presence of EspA polypeptide in the sample is indicative ofinfection by enteropathogenic E. coli.
 14. The method of claim 10,wherein the presence of EspA polypeptide in the sample is indicative ofinfection by enterohemorrhagic E. coli.
 15. A method of immunizing ahost susceptible to disease caused by an EspA-producing organism,comprising: a) administering to the host an EspA polypeptide of claim 1;and b) inducing a protective immune response to EspA in the host. 16.The method of claim 15, wherein the EspA-producing organism is E. coli.17. The method of claim 16, wherein the EspA-producing E. coli. isenteropathogenic E. coli.
 18. The method of claim 16, wherein theEspA-producing E. coli. is enterohemorrhagic E. coli.
 19. A method ofameliorating disease caused by EspA-producing organism, comprising: a)immunizing a host with the polypeptide of claim 1; and b) inducing animmune response in the host to the EspA polypeptide, therebyameliorating disease caused by infection of the host by EspA-producingorganism.
 20. The method of claim 19, wherein the EspA-producingorganism is E. coli.
 21. The method of claim 19, wherein theEspA-producing E. coli. is enteropathogenic E. coli.
 22. The method ofclaim 19, wherein the EspA-producing E. coli. is enterohemorrhagic E.coli.
 23. A method for detecting a polynucleotide in a sample,comprising: a) contacting a sample suspected of containing espApolynucleotide with a nucleic acid probe that hybridizes to thepolynucleotide of claim 2; and b) detecting hybridization of the probewith the polynucleotide, wherein the detection of hybridization isindicative of espA polynucleotide in the sample.
 24. A method forproducing a recombinant espA polynucleotide, comprising: inserting anucleic acid encoding a selectable marker into the polynucleotide ofclaim 2, such that the resulting polynucleotide encodes a recombinantEspA polypeptide containing the selectable marker.
 25. A polynucleotideproduced by the method of claim
 24. 26. A host cell containing thepolynucleotide of claim
 25. 27. A method for producing a recombinantEspA polypeptide, comprising: a) growing a host cell containing apolynucleotide encoding a EspA polypeptide of claim 1 under conditionswhich allow expression of EspA polypeptide; and b) isolating thepolypeptide.
 28. A method to identify a compound that affects bacterialtype III secretion, comprising: a) introducing the polynucleotide ofclaim 5 into bacteria having a bacterial type III secretion system; b)growing the bacteria under conditions which allow expression of thepolypeptide encoded by the polynucleotide; c) contacting the bacteriawith a candidate compound; and d) measuring secretion of thepolypeptide, and thereby identifying a compound that affects type IIIsecretion.
 29. A method for producing a nonpathogenic organism,comprising: a) generating a mutation in a polynucleotide encoding a EspApolypeptide of claim 1; b) inserting a nucleic acid sequence encoding aselectable marker into the site of the mutation; c) introducing themutated espA polynucleotide of step b) into a chromosomal espA gene ofan organism to produce a mutation in the chromosomal espA gene; and d)selecting organisms having the mutation.
 30. The method of claim 29,wherein the nucleic acid sequence encoding a selectable marker encodesresistance to kanamycin.
 31. The method of claim 29, wherein theorganism is E. coli.
 32. An organism with a mutated espA gene producedby the method of claim
 29. 33. A kit useful for the detection of a EspApolypeptide of claim 1, comprising carrier means being compartmentalizedto receive in close confinement therein one or more containerscomprising a container containing an antibody which binds to EspApolypeptide.
 34. The kit of claim 33, wherein the antibody is detectablylabeled.
 35. The kit of claim 34, wherein the label is selected from thegroup consisting of radioisotope, a bioluminescent compound, achemiluminescent compound, a fluorescent compound, a metal chelate, andan enzyme.
 36. A kit useful for the detection of an espA polynucleotideof claim 2, comprising carrier means being compartmentalized to receivein close confinement therein one or more containers comprising acontainer containing the nucleic acid probe that hybridizes to espApolynucleotide.
 37. The kit of claim 36, wherein the probe is detectablylabeled.
 38. The kit of claim 37, wherein the label is selected from thegroup consisting of radioisotope, a bioluminescent compound, achemiluminescent compound, a fluorescent compound, a metal chelate, andan enzyme.
 39. A method of producing a fusion protein comprising: a)growing a host cell containing a polynucleotide of claim 2 operablylinked to a polynucleotide encoding a polypeptide or peptide of interestunder conditions which allow expression and secretion of the fusionprotein; and b) isolating the fusion protein.